Methods and compositions for the control of coccidiosis

ABSTRACT

Methods are provided for the sporulation, sterilization and storage of coccidial oocyst which are characterized by an absence of the highly toxic chemical potassium dichromate. Also provided are compositions containing sporulated oocysts which are free of potassium dichromate.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of pending application Ser. No.10/005,510, filed Nov. 8, 2001, which claims the benefit of U.S.Provisional Application No. 60/246,847, filed Nov. 8, 2000, both ofwhich are hereby incorporated by reference in their entirety.

BACKGROUND

[0002] Coccidiosis is a disease of various animals in which theintestinal mucosa is invaded and damaged by a protozoa of the subclassCoccidia. The economic effects of coccidiosis can be especially severein the poultry industry where intensive housing of birds favors thespread of the disease. Infection by coccidial protozoa is, for the mostpart, species specific. Numerous species, however, can infect a singlehost. For example, there are seven species of coccidial protozoa whichinfect chickens, six of which are considered to be moderately toseverely pathogenic.

[0003] The life cycle of the coccidial parasite is complex. For example,protozoa of the genera Eimeria, Isospora, Cystoisospora, orCryptosporidium typically only require a single host to complete theirlife cycle, although Cystoisospora may utilize an intermediate host.Under natural conditions, the life cycle begins with the ingestion ofsporulated oocysts from the environment. When sporulated oocysts areingested by a susceptible animal, the wall of the sporulated oocyst isbroken in order to release the sporocysts inside. In poultry, therelease of the sporocyst is the result of mechanical disruption of thesporulated oocyst in the gizzard. Within the sporocysts, are thesporozoites which are the infective stage of the organism. In poultry,the breakdown of the sporocyst coat and release of the sporozoites isaccomplished biochemically through the action of chymotrypsin and bilesalts in the small intestine. Once released, the sporozoites invade theintestinal mucosa or epithelial cells in other locations. The site ofinfection is characteristic of the species involved. For example, in thegenus Eimeria, E. tenella is localized in the ceca; E. necatrix is foundin the anterior and middle portions of the small intestine; E.acervulina and E. praecox occur in the upper half of the smallintestine; E. brunetti occurs in the lower small intestine, rectum,ceca, and cloaca; E. mitis is found in the lower small intestine, whileE. maxima can be found in any of these physiological locations.

[0004] Once inside the host animals' cells, sporozoites develop intomultinucleate meronts, also called schizonts. Each nucleus of the merontdevelops into an infective body called a merozoite which enters newcells and repeats the process. After a variable number of asexualgenerations, merozoites develop into either microgametocytes ormacrogametes. Microgametocytes develop into many microgametes which, inturn, fertilize the macrogametes. A resistant coat then forms around theresulting zygotes. The encysted zygotes are called oocysts and are shedunsporulated in the feces. Infected birds may shed oocysts in the fecesfor days or weeks. Under proper conditions of temperature and moisture,the oocysts become infective through the process of sporulation.Susceptible birds then ingest the sporulated oocysts through normalpecking activities or ground/litter foraging and the cycle repeatsitself. Ingestion of viable, sporulated oocysts is the only naturalmeans of infection.

[0005] Infection with coccidial protozoa results in immunity so that theincidence of the disease decreases over time as members of the flockbecome immune. This self-limiting nature of coccidial infections iswidely known in chickens and other poultry. The immunity conferred,however, is species specific such that introduction of another speciesof coccidial protozoa will result in a new disease outbreak.

[0006] The oocyst wall of coccidial protozoa provides a highly effectivebarrier for oocyst survival. Oocysts may survive for many weeks outsidethe host. In the laboratory, intact oocysts are resistant to extremes inpH, detergents, proteolytic, glycolytic, and lipolytic enzymes,mechanical disruption, and chemicals such as sodium hypochlorite anddichromate.

[0007] Two methods are currently used to control coccidiosis in poultry.The first involves control by chemotherapy. Numerous drugs are availablefor the control of coccidiosis in poultry. Because of the number ofspecies which cause the disease, very few drugs are efficacious againstall species, although a single drug may be efficacious against severalspecies. In modern broiler chicken production, for example,administration of drugs to control coccidiosis is routine. The expensefor preventative medication against coccidiosis represents a significantcost of production.

[0008] Two programs of drug administration are commonly used in thedomestic poultry industry. The simplest is the continuous use of asingle drug from day one following hatching until slaughter. The secondprogram is to use shuttle or dual drug program which involves the use oftwo different drugs, one administered in the “starter” ration and asecond drug administered in the “grower” ration. This second method isoften preferred as a method to minimize development of drug resistantstrains of Coccidia. Using either method, drugs used are typicallyrotated two to three times per year in order to minimize the developmentof resistant strains.

[0009] The development of drug resistance by Coccidia is a seriouslimitation on the effectiveness of chemotherapy to control the disease.Surveys in the United States, South America and Europe have revealedwidespread drug resistance in Coccidia. Since drug resistance is agenetic phenomenon, once established, drug resistance can remain in thepopulation for many years until reduced by natural selection pressureand genetic drift.

[0010] The use of drugs in animals used for food production is alsocoming under increasing scrutiny by the public. Consumers areincreasingly concerned with the possibility of drug residues in food.This creates pressure in the poultry industry to reduce the use of drugsto control coccidiosis.

[0011] Vaccination of birds against coccidiosis is an alternative tochemotherapy. An advantage of vaccination is that it can greatly reduceor eliminate the need to administer anti-coccidial drugs, thus reducingdrug costs to poultry producers, preventing the development ofdrug-resistant strains, and lessening consumer concerns about drugresidues.

[0012] Numerous methods have been developed to immunize poultry againstcoccidial protozoa. The successful methods have all been based on theadministration of live protozoa, either fully virulent strains orattenuated strains. The most common route of administration is oral,although other routes have been used. Edgar, U.S. Pat. No. 3,147,186,teaches vaccination of chickens by oral administration either directlyinto the mouth or via the feed or water of viable E. tenella sporulatedoocysts. Davis et al., U.S. Pat. No. 4,544,548, teaches a method ofvaccination by continuous administration of low numbers of sporulatedoocysts, with or without simultaneous administration of anti-coccidialdrugs.

[0013] Oral administration of attenuated strains of sporocysts has alsobeen utilized to confer immunity against coccidiosis. Shirley, U.S. Pat.No. 4,438,097; McDonald, U.S. Pat. No. 5,055,292; and Schmatz et al.,PCT publication No. WO 94/16725. An alternative to attenuation isdisclosed in Jenkins et al., Avian Dis., 37(1):74-82 (1993), whichteaches the oral administration of sporozoites that have been treatedwith gamma radiation to prevent merogonic development.

[0014] Parenteral routes of vaccination have included subcutaneous orintraperitoneal injection of excysted sporozoites, Bhogal, U.S. Pat. No.4,808,404; Bhogal et al., U.S. Pat. No. 5,068,104, and intra ovoinjection of either oocysts or sporocysts, Evans et al., PCT publicationNo. WO 96/40233; Watkins et al., Poul. Sci., 74(10):1597-602 (1995).Sharma, J. Parasitol., 50(4):509-517 (1964), reported unsuccessfulimmunization trials involving intravenous, intraperitoneal,intramuscular, or subcutaneous injection of either viable oocysts or amixture of oocysts, sporocysts and sporozoites. Thaxton, U.S. Pat. No.5,311,841, teaches a method of vaccination against Coccidia byadministration of oocysts or sporozoites to newly hatched chicks by yolksac injection.

[0015] Regardless of the route of administration, procedures for theproduction of coccidiosis vaccines are quite similar. Briefly, coccidialprotozoa are produced by infecting host animals with a single species ofcoccidial protozoa. These “seed stocks” are often clonal in nature, thatis, derived from a single organism in order to insure the presence ofonly the species of interest. Seed stocks may be wild type, that is,isolated from the field, or they may be precocious or attenuatedstrains. The protozoa are then allowed to undergo replication in thehost, after which, protozoa are collected from the animals, usually fromthe excreta. The use of attenuated strains typically results in fewershed oocysts from the host animal. The protozoa are then separated fromthe excreta by well known techniques such as salt floatation andcentrifugation. At the time of collection, the protozoa are at thenon-infective oocyst stage of the life cycle. In order to becomeinfective, and therefore useful for vaccines, the oocysts must beinduced to undergo sporulation. In members of the genus Eimeria,sporulation typically involves the incubating the oocysts in a 1% to 4%aqueous solution of potassium dichromate at 19° C. to 37° C. withconstant aeration. Data on oxygen consumption are conflicting, withSmith and Wilson (J. Parasitol. 30:295-302, 1944) reporting increasedoxygen consumption for E. tenella and Wilson and Fairbairn (J.Protozool. 8:410-416, 1961) reporting no change in oxygen consumptionfor E. acervulina. Sporulation is usually complete within 12 to 24 hoursdepending on the temperature used. Monitoring of the sporulation processis accomplished by microscopic examination of the protozoa. Storagecompositions found in the prior art typically include an aqueoussolution of potassium dichromate. The sporulated oocysts are usuallystored in 1 to 4% aqueous solution of potassium dichromate to preventbacterial growth, however, other storage media have been used.

[0016] Current vaccines available for the prevention of coccidiosistypically contain a 2.5% weight to volume solution and containapproximately 1,600 oocyts per dose (400 sporulated oocysts representingfour different species). The current commercially available vaccinescontain from about 1.6×10⁻² μg of potassium dichromate per oocyst toabout 0.16 μg of potassium dichromate per oocyst.

[0017] Although widely used for sporulation and storage, potassiumdichromate has several properties which make its elimination frombiologicals highly desirable. Potassium dichromate is a strong oxidizerand has been reported to affect the respiratory system, liver, kidneys,eyes, skin and blood. It is a known carcinogen and upon disposal isregarded as a hazardous waste. Because of its high toxicity, compoundscontaining potassium dichromate are particularly unsuitable forparenteral administration. Thus, it would be highly advantageous toeliminate potassium dichromate from the production and storage ofmaterials to be administered to animals, especially food animals.

SUMMARY

[0018] Coccidiosis is a disease of animals which has a significanteconomic impact, especially in the poultry industry. In many poultryoperations, birds are vaccinated against coccidiosis using vaccinescontaining sporulated oocysts. Present methods for the sporulation andstorage of coccidial oocysts use the highly toxic chemical potassiumdichromate. The present invention provides a method for the sporulation,sterilization and storage of coccidial oocysts, without the use ofpotassium dichromate. The present invention also provides for vaccinecompositions that can be administered to animals, particularly from theclass Aves, and more particularly poultry, said vaccines beingcharacterized as substantially free of potassium dichromate, both interms of their production and storage. Due to its high toxicity, theelimination of potassium dichromate is particularly desirable in theproduction and storage of compositions to be administered to animals,and in particular to food producing animals. As the vaccine compositionsof the present invention are sterile, the vaccine compositions can beadministered to animals, through various routes, including, but notlimited to orally, e.g, by addition to food or water; topically, e.g.,spraying; parenteral routes, e.g. subcutaneous, intramuscular orintraperitoneal injection; per os or via intra-yolk sac injection.

[0019] As used herein, the term substantially free of alkali metaldichromate indicates that no alkali metal dichromate is added to thecomposition during production, including the sporulation and storage ofsaid composition. Furthermore, as used herein, the term substantiallyfree of potassium dichromate indicates that no potassium dichromate isadded to the composition during production, including the sporulationand storage of said composition.

[0020] The present invention provides a method for isolating oocysts,concentrating oocysts, sporulating oocysts, isolating sporulatedoocysts, sterilizing sporulated oocysts and storage of sporulatedoocysts. More particularly, the present invention provides a method forsporulating oocysts, isolating sporulated oocysts, sterilizingsporulated oocysts and storage of sporulated oocysts.

[0021] Among the several aspects of the invention, is provided a vaccinefor the prevention and/or control of coccidiosis comprising viablesporulated oocysts of at least one species of protozoa known to causecoccidiosis, wherein the composition is sterile and characterized assubstantially free of potassium dichromate.

[0022] Another aspect provides a vaccine for the prevention and/orcontrol of coccidiosis comprising an aqueous diluent and viablesporulated oocysts of at least one species of protozoa known to causecoccidiosis wherein the vaccine is sterile and characterized assubstantially free of potassium dichromate.

[0023] Another aspect of the invention provides a method of isolatingand concentrating oocysts to prepare the oocysts for sporulationcomprising a novel combination of isolation, cleansing, andconcentration steps. The methods of the instant invention may be usedindividually or in combination with one another.

[0024] The method of isolating and concentrating oocysts comprisessieving a manure slurry known to contain oocysts. The method furthercomprises concentrating the oocysts by centrifugal-based separation. Themethod further comprises a flotation step to further cleanse and isolatethe oocysts. The method further comprises centrifugal-based separation,followed by a flotation step, followed by another application ofcentrifugal-based separation.

[0025] Another aspect provides a method for isolating oocysts comprisingcollecting feces that contains oocysts known to cause coccidiosis. Thefeces are contacted with an aqueous medium, and unwanted fecal matter isseparated from the oocysts. The oocysts are subjected tocentrifugal-based separation and the solid oocyst-containing fraction iscollected and suspended in a flotation solution. The oocysts are allowedto separate from the solids, and the flotation medium is removed fromthe oocysts by tangential flow filtration.

[0026] A further aspect provides a method for inducing the sporulationof oocysts comprising incubating viable oocysts of at least one speciesof protozoa known to cause coccidiosis in an aqueous medium whereindissolved oxygen concentration is maintained from about 30% to about 80%of saturation. Temperature is controlled from a temperature whichprevents substantial freezing up to about 43° C. An oxidizing agent,other than potassium dichromate, is added at a sporulation inducingconcentration to form a sporulation medium; and the sporulation mediumis incubated to form sporulated oocysts.

[0027] Another aspect provides a method for inducing the sporulation ofoocysts comprising incubating viable oocysts of at least one species ofprotozoa known to cause coccidiosis in an aqueous medium whereindissolved oxygen concentration is maintained at at least about 50% ofsaturation. Temperature is controlled from a temperature which preventssubstantial freezing up to 43° C. An oxidizing agent, other than analkali metal dichromate, soluble dichromate moieties, dichromate ions,or potassium dichromate, to form a sporulation medium; and wherein theoxidizing agent is at a sporulation inducing concentration. Thesporulation medium is then incubated to form sporulated oocysts; thesporulated oocysts are then separated from the sporulation medium; thesporulated oocysts are then sterilized with a chemical disinfectant; thechemical disinfectant is removed by tangential flow filtration. Thesporulated oocysts may then be stored in a diluent substantially free ofpotassium dichromate as it has been discovered that an oxidizing agent,other than dissolved oxygen available in sterile water, is not necessaryto preserve viability for useful periods of time. However, the use of anoxidizing agent may increase longevity of the vaccine, and thus, may beused as part of the instant invention.

[0028] Another aspect provides a method for sporulating oocystscomprising incubating oocysts of at least one species of protozoa knownto cause coccidiosis in an aqueous sporulation medium, and separatingthe oocysts by tangential flow filtration from the sporulation medium.

[0029] Yet another aspect provides a method for sterilizing oocystscomprising contacting oocysts of at least one species of protozoa knownto cause coccidiosis with a sterilization medium, and removing thesterilization medium from the oocysts by tangential flow filtration.

[0030] Still a further aspect provides, a method for monitoringsporulation of oocysts comprising, incubating viable oocysts in a mediumunder sporulation inducing conditions, and monitoring the medium duringthe incubation for a change in pH, or a change in the combination ofdissolved oxygen and pH, the change being characteristic of sporulation.

[0031] Yet another aspect provides, a kit for the prevention and/orcontrol of coccidiosis comprising a vaccine comprising sterile, viable,sporulated oocysts of at least one species of protozoa known to causecoccidiosis, the vaccine being characterized as substantially free ofpotassium dichromate; and instructions for administering the compositionto an animal. In another embodiment, the kit further comprises adiluent, which may be sterile, and instructions for mixing the oocystswith the diluent to form a mixture and for administering the mixture toan animal.

[0032] Another aspect provides, a composition for the storage ofsporulated oocysts comprising an aqueous diluent and a bactericide, thecomposition being sterile and being characterized as substantially freeof potassium dichromate. In yet another embodiment the aqueous diluentcomprises water. In a further embodiment the aqueous diluent comprisesdomestic water. In a further embodiment the aqueous diluent comprisesfrom about 0.1×to about 1× phosphate buffered saline (PBS) and fromabout 0 to about 30 μg/ml gentamicin.

[0033] A further aspect provides, a method for storing sporulatedoocysts comprising obtaining sterile, sporulated oocysts of at least onespecies of protozoa known to cause coccidiosis and placing thesporulated oocysts in a composition comprising an aqueous diluent and abactericide, the composition being sterile and being characterized assubstantially free of added potassium dichromate.

[0034] Yet another aspect provides, a method for storing sporulatedoocysts comprising obtaining sterile, sporulated oocysts of at least onespecies of protozoa known to cause coccidiosis; placing the sporulatedoocysts in a sterile composition comprising 0.5×PBS and about 30 μg/mlgentamicin, the composition being characterized as substantially free ofpotassium dichromate; and storing the composition containing thesporulated oocysts at less than about 10° C., preferably between about1° C. to about 8° C., more preferably from about 3° C. to about 6° C.,and most preferably, about 4° C.

[0035] In yet another aspect, the invention provides a combination ofspecies of oocysts from the genus Eimeria comprising a minimumimmunizing dose of oocysts.

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] These and other features, aspects, and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims and accompanying figures where:

[0037]FIG. 1 shows a graph of percent saturation of dissolved oxygen (A)and pH (B) versus time of the sporulation medium during a successfulsporulation when percent saturation of dissolved oxygen is notcontrolled.

[0038]FIG. 2 shows a graph of percent saturation of dissolved oxygen (A)and pH (B) versus time of the sporulation medium during a successfulsporulation in which the rise in pH was preceded by a drop in pH whenpercent saturation of dissolved oxygen and pH is not controlled.

[0039]FIG. 3 shows the percent viable oocysts recovered (PVOR) versusstorage time for oocytes stored under the conditions indicated. Allstorage medium contained P. acnes and oocysts were sterilized by 5%NaOCl.

[0040]FIG. 4 shows the percent viable oocysts recovered (PVOR) versusstorage time for oocytes stored under the conditions indicated. Allstorage medium contained P. acnes and oocysts were sterilized by 2%NaOCl.

[0041]FIG. 5 shows a flow diagram of the process used to produce oralcoccidiosis vaccine. The flow chart is divided into four suites: (1)Challenge Suite; (2) Purification Suite; (3) Sporulation Suite; and (4)Storage Suite. Steps 1 through 34 are described for illustrativepurposes as follows:

[0042]1. Feed given to the birds

[0043]2. Water given to the birds

[0044]3A. Manure is harvested after birds begin shedding oocysts

[0045]3B. Manure is discarded prior to when birds shed oocysts

[0046]4. Water is added to a slurry tank containing the manure

[0047]5. The slurry is sieved

[0048]6. Solid waste from sieving is discarded

[0049]7. Filtrate collection transferred to and then separated bycentrifugal-based separation

[0050]8. Liquid waste is discarded while moist solid is retained

[0051]9. Moist solid is transferred to mix tank

[0052]10. High fructose corn syrup is added to tank

[0053]11. Water is added (if needed) to adjust specific gravity

[0054]12. Suspension is transferred for separation

[0055]13. Solid wastes are discarded

[0056]14. Liquid phase is transferred to mix tank

[0057]15. Water is added to mix tank

[0058]16. Diluted suspension is transferred to and then centrifuged

[0059]17. Liquid waste is discarded

[0060]18. Solid transferred to blend vessel

[0061]19. Water is added to blend vessel

[0062]20. Diluted suspension is deposited in holding container thenlater transferred to sporulation vessel

[0063]21. An oxidizing agent is added to the sporulation medium

[0064]22. Sporulated oocysts transferred from sporulation vessel toseparation device

[0065]23. Water is added to sporulated oocysts suspension

[0066]24. Waste water from separation is removed and discarded

[0067]25. Sporulated oocysts, now separated from sporulation medium, aretransferred to sterile filtration unit

[0068]26. Addition of disinfecting agent

[0069]27. Addition of sterile water

[0070]28. Waste water and disinfecting agent are discarded

[0071]29. Transfer to holding vessel

[0072]30. Addition of buffer

[0073]31. Addition of bactericide

[0074]32. Transfer to blending vessel where buffer and post-challengeperformance improvement composition are blended

[0075]33. Vialing

[0076]34. To kit or to market

DETAILED DESCRIPTION

[0077] The following detailed description is provided to aid thoseskilled in the art in practicing the present invention. Even so, thisdetailed description should not be construed to unduly limit the presentinvention as modifications and variations in the embodiments discussedherein can be made by those of ordinary skill in the art withoutdeparting from the spirit or scope of the present inventive discovery.

[0078] All publications, patents, patent applications, databases andother references cited in this application are herein incorporated byreference in their entirety as if each individual publication, patent,patent application, database or other reference were specifically andindividually indicated to be incorporated by reference.

[0079] As used herein, the term “domestic water” refers to available(potable) tap water.

[0080] The present invention provides for the substantial elimination ofadded potassium dichromate in the production of vaccines used for theprevention of coccidiosis in animals, and in particularly poultry. Morespecifically, the composition of the invention is a novel vaccine readyfor administration. The composition of the invention herein, in apreferred embodiment, is substantially free of alkali metal dichromates,soluble dichromates, dichromate ions, and potassium dichromate when themethods disclosed herein are practiced. Any alkali metal dichromates,soluble dichromates, dichromate ions, and potassium dichromate presentin the compositions described herein or present in the compositions madeby the methods described herein would be residual and, in a preferredembodiment, would not be present due to affirmative addition. Inaddition, the present invention provides methods for the sporulation ofcoccidial protozoa, the production of sterile preparations containingviable sporulated oocysts that are substantially free of potassiumdichromate, and compositions and methods for storing sporulated oocysts.

[0081] Furthermore, the invention provides a method for the monitoringof the sporulation process that does not require microscopic examinationof the oocysts. Furthermore, the invention provides for kits containinga composition used for the prevention of coccidiosis in animals, and inparticularly poultry characterized as substantially free of potassiumdichromate.

[0082] Oocysts used in the practice of the present invention can beobtained from a variety of sources. For example, oocysts can be obtainedby the inoculation of host animals with coccidial protozoa of a singlespecies or with a mixture of species. The coccidial protozoa used can beclonal in nature, that is derived from a single progenitor, orpolyclonal. The oocysts of the present composition are derived from wildtype oocysts. Inoculation can be by any means which will allow for thereplication of the protozoa in the host animal. The most common route ofinoculation is per os, but other suitable routes may be used. Ifadministered per os, the protozoa are preferably at the sporulatedoocyst stage. Administration can be by gavage or through the feed and/orwater. Inoculation can also be accomplished by exposing host animals toenvironments contaminated with coccidial protozoa. Alternatively,oocysts can be obtained from animals with naturally occurringinfections.

[0083] The present invention provides a method for the sporulation ofoocysts comprising first orally inoculating host animals with Eimeriaspecies. Manure from the innoculated animals is then collected from theinoculated host animals. Oocysts are separated from the manure using acombination of isolation techniques, including sieving, centrifugationand density flotation. The isolated oocysts are then sporulated undercertain defined conditions controlling factors such as temperature,percent saturation of dissolved oxygen, pH, and agitation in asporulation medium. The sporulated oocysts are then separated from thesporulation medium and disinfected. After, the sporulated oocysts havebeen separated from the sporulation medium and disinfected they arecombined with a diluent or a diluent and a buffer to form a vaccine. Apost-challenge performance improvement composition may also then beadded. Such a composition ameliorates a decrease in post-challengeperformance. The amelioration may be seen in factors such as bursalgrowth and appearance. For illustrative purposes only, a preferredmethod is described graphically in the form of a flow sheet in FIG. 5.

[0084] Furthermore, the invention provides for a method of monitoringsporulation without the need for microscopic observations. Microscopicobservation requires sampling and technician labor. The elimination ofmicroscopic observation provides a more efficient method for monitoringsporulation. The method of the invention for monitoring sporulationcomprises monitoring oxygen consumption, as evidenced by a change indissolved oxygen content, and monitoring pH. It has been discovered thatsporulation results in an increase in oxygen consumption, as evidencedby a decrease in dissolved oxygen in a sporulation medium, and thatsporulation results in an increase in pH. See FIGS. 1 and 2. Sporulationrates may be monitored by monitoring the volume and flow rate of anoxygen containing gas added to control percent saturation of dissolvedoxygen and also may be monitored by monitoring the acid or base addedneeded to maintain a desired pH. Thus, the decrease in dissolved oxygenis a differential decrease sufficient to trigger the addition, by acontrolled device, to supply more oxygen. In addition, the decrease inpH is also a differential decrease that is sufficient to trigger theaddition of an acid or a base.

[0085] Methods of Isolation, Concentration, and Purification

[0086] In general, a number of different methods of preparing oocystsfor sporulation are known in the art. Any one or combination of suchmethods may be used prior to sporulation. However, a preferred method isset out below. A number of well known processes are set forth to assistone skilled in the art to practice the invention in its differentembodiments.

[0087] To begin, once host animals begin shedding the organism, theprotozoa can be collected. Most commonly, protozoa are collected fromthe feces, but they can also be collected from intestinal contentsand/or scrapings as well as contaminated bedding (see FIG. 5A,“Challenge Suite”). Once collected, the oocysts are preferably isolatedfrom the extraneous fecal material as decreasing the fecal content in anoocyst suspension increases the number of oocysts that will sporulate(Smith and Ruff, Poultry Sci. 54:2083, 1975). As will be describedbelow, a preferred method for isolating oocysts is by sieving (see FIG.5A, step 5). However, several methods for isolating protozoa are knownin the art and may be used in practicing the present invention. Asummary of these isolation methods are summarized herein followed by adescription of the preferred method. Several methods described hereinprocess the collected manure to a point wherein sporulation may thenbegin. Others require further processing, such as further isolation orcleansing. This further processing may be accomplished by utilizing themethods and techniques described herein or a combination thereof.

[0088] A review of several methods for the isolation of oocysts can befound in Ryler et al. (Parasitology 73:311-326, 1976). In one method,described in U.S. Pat. No. 3,147,186, oocysts are only crudely isolatedfollowing the addition of the oxidizing agent potassium dichromate. Inthis method, the moist droppings of host animals are directly mixed withan aqueous solution containing between one and four percent potassiumchromate, preferably 2.5% or, less preferably, water, so that asuspension of thin consistency is obtained. The method indicates that aconcentration of at least about one to four percent potassium chromatesolution is necessary to obtain adequate oocyst sporulation. Largerinsoluble debris, such as feathers and partially digested or undigestedfeed, is removed. Removal can be done conventionally by filtering thesuspension through a mesh screen. The suspension is then allowed tostand for about five minutes to allow heavier coarser particles ofdebris that passed through the screen to settle to the bottom of theholding container. The supernatant liquid containing the oocysts is thenremoved. The sporulated oocysts are viable for up to about 18 hours.

[0089] Another method for separating oocysts from droppings comprisesflotation using solutions of sufficient specific gravity, typicallyhaving a specific gravity of about 1.2, so that oocysts float to the topof the suspension. Generally these solutions are made up of water towhich a sugar (e.g. sucrose), ZnSO₄, or NaCl has been added to increasethe specific gravity to the desired value. Useful solutions includesolutions comprising 58% (w/v) sucrose, 37% (w/v) ZnSO₄ □ 7H₂O andsaturated NaCl solutions, which all have a specific gravity from about1.09 to about 1.2. Other solutions which have a comparable specificgravity and are not harmful to the oocysts can also be used.

[0090] In the flotation method of isolation, a preliminary step offiltering diluted collected manure through, e.g., gauze, a sieve orcheesecloth to remove large particles of undesired fecal matter may beincluded. After mixing harvested oocysts with the flotation solution,the oocyst slurry may be centrifuged and the oocyst removed from thesurface layer of the supernatant. The centrifugation step may berepeated several times to further purify the oocysts by resuspending thecaptured supernatant in a flotation medium having a specific gravitysimilar to that used in previous centrifugation steps and centrifugedagain. This step may be repeated until the desired level of purity isreached.

[0091] Another method for isolation of oocysts available in the artcomprises gradient centrifugation. The gradient used can bediscontinuous or continuous. An example of a typical gradient forcoccidial oocysts is 0-50% sucrose. In this method the materialcontaining the oocysts is placed on top of the gradient and the oocystcontaining material is then centrifuged along with the gradient.Following centrifugation, the layer containing the oocysts is recovered.The process may be repeated in order to increase the purity of theresulting oocyst preparation. As with flotation, this method ispreferably preceded by filtration of the collected manure.

[0092] Additional methods of oocyst isolation include, the use of glassbead columns (Ryler et al., Parasitology, 73:311-326, 1976) and thebicarbonate ether method (Smith and Ruff, Poultry Sci. 54:2081-2086,1975). In the glass column method, the aqueous suspension of fecalmatter is added to a mixture of glass beads and a detergent, for example5% Tween 80. The mixture is then applied to a column of glass beads andthe oocysts are allowed to flow through while much of the undesiredfecal matter is retained in the column. The effluent may then beconcentrated by centrifugation.

[0093] In the bicarbonate ether method, the feces from infected chickensis strained, through cheese cloth for example, and the liquid fractionis captured while the solid fraction is discarded. The liquid fractionis then concentrated by centrifugation. The solid fraction is recoveredand the supernatant is discarded. The recovered solid fraction is thenresuspended in a solution of 1% sodium bicarbonate. To the resuspendedsolid fraction, now in suspension, is then added ether in a volumeapproximately equal to the volume of 1% solution of sodium bicarbonate.The mixture is then centrifuged. The debris plug and supernatant isdiscarded while the sediment is washed by resuspension in water. Thissuspension is then centrifuged and the supernatant discarded. Thesediment is then recovered for use. (Smith and Ruff, Poultry Sci.54:2081-2086, 1975).

[0094] The various methods for isolating, concentrating, and purifyingoocysts describe above may be used in combination with one another or incombination with the preferred embodiments of the instant invention.Regardless of the methods used, the greater the isolation,concentration, and purification the greater percent sporulation duringthe sporulation suite (see FIG. 5, “Sporulation Suite”). Therefore, ithas been discovered that the following methods of isolation,concentration, purification, sporulation, sterilization, and storageprovide a novel and improved method for the production of spotulatedoocysts.

[0095] One aspect of the method of the instant invention comprisescollecting manure from host animals wherein said manure contains oocystsknown to cause coccidiosis; diluting said manure in an aqueous medium tocreate a slurry; separating unwanted fecal matter from said slurry andcollecting the aqueous fraction containing oocysts; subjecting saidaqueous fraction to solid/liquid phase centrifugal-based separation andcollecting the solid phase; combining a dense aqueous liquid with saidcollected solid phase wherein said dense liquid has a density greaterthan about 1.09 g/ml and wherein the oocysts are buoyant; subjecting thecombination of said dense aqueous liquid and collected solid phase tocentrifugation and collecting the dense liquid fraction containingoocysts, diluting said dense liquid fraction to a specific gravitywherein the oocysts are no longer buoyant; separating oocyst solids fromsaid diluted liquid fraction by centrifugal-based separation andre-collecting the solid phase.

[0096] In another aspect the method for isolating oocysts comprisescollecting manure from host animals wherein said manure contains oocystsknown to cause coccidiosis; diluting said manure in an aqueous medium tocreate a slurry; separating unwanted fecal matter from said slurry andcollecting the aqueous fraction containing oocysts; subjecting saidaqueous fraction to solid/liquid phase centrifugal-based separation bymeans of a hydrocyclone.

[0097] In yet another aspect, the method for isolating oocysts comprisescollecting manure from host animals wherein said manure contains oocystsknown to cause coccidiosis; diluting said manure in an aqueous medium tocreate a slurry; separating unwanted fecal matter from said slurry andcollecting the aqueous fraction containing oocysts; subjecting saidaqueous fraction to solid/liquid phase centrifugal-based separation andcollecting the solid phase; combining a dense aqueous liquid with saidcollected solid phase wherein said dense liquid has a density greaterthan about 1.09 g/ml and wherein the oocysts are buoyant; subjecting thecombination of said dense aqueous liquid and collected solid phase tocentrifugation and collecting the dense liquid fraction containingoocysts, diluting said dense liquid fraction to a specific gravitywherein the oocysts are no longer buoyant; separating oocyst solids fromsaid liquid phase by means of a hydrocyclone and re-collecting the solidphase.

[0098] A further aspect of the methods provided herein describes amethod for sporulating oocysts comprising introducing into an aqueoussporulation medium oocysts of at least one species of protozoa known tocause coccidiosis; incubating said oocysts in said aqueous sporulationmedium; and introducing an oxidizing agent into said medium at a ratesufficient to maintaining the dissolved oxygen content of the medium atat least 30% of saturation; said medium containing less than about 0.8%by weight alkali metal dichromate during incubation of said oocysts.

[0099] Another aspect of the methods provided herein describes a methodfor sporulating oocysts comprising introducing into an aqueoussporulation medium oocysts of at least one species of protozoa known tocause coccidiosis; incubating said oocysts in said aqueous sporulationmedium; and introducing an oxidizing agent into said medium at a ratesufficient to maintaining the dissolved oxygen content of the medium atbetween about 30% and about 80% of saturation; said medium containingless than about 0.8% by weight alkali metal dichromate during incubationof said oocysts.

[0100] In another aspect of the methods provided herein is described amethod for separating sporulated oocysts from a sporulation medium;sterilizing sporulated sporocysts by contacting said sporulated oocystswith a chemical disinfectant; and storing said sporulated oocysts in asterile diluent, wherein said diluent contains less than about 0.8% byweight alkali metal dichromate.

[0101] In another aspect of the methods described herein is described amethod for inducing sporulation of oocysts comprising introducing intoan aqueous sporulation medium oocysts of at least one species ofprotozoa known to cause coccidiosis; incubating said oocysts in saidaqueous sporulation medium; and introducing an oxidizing agent having astandard reduction potential of at least about 0.5 V at a ratesufficient to maintain the oxidation potential of said medium equivalentto the oxidation potential of a medium containing dissolved molecularoxygen in concentration of at least 30% of saturation; said mediumcontaining less than about 0.8% by weight alkali metal dichromate duringincubation of said oocysts.

[0102] In yet another aspect of the instant invention, there isdescribed a method for monitoring sporulation comprising incubatingviable oocysts in an aqueous sporulation medium; and during incubation,monitoring said medium to detect a change in at least one of thefollowing parameters: (i) dissolved oxygen content; (ii) pH; (iii) rateof introduction of oxidizing agent into said medium; (iv) flow rate ofacid or base into said medium.

[0103] In another aspect of the instant invention, there is provided acomposition for the storage of sporulated oocysts comprising 0.5×PBS;and about 30 μg/ml gentamicin, wherein said composition is characterizedas substantially free of alkali metal dichromate, and furthercharacterized in that oocysts in contact with said composition decreasein viability no more than about 20% over a period of at least about 26weeks at about 5° C.

[0104] In a further aspect of the instant invention, a method of storingsporulated oocysts is provided that comprises contacting sporulatedoocysts with the storage composition described above.

[0105] In yet but another aspect, the instant invention provides for akit comprising a composition containing, sterile, viable, sporulatedoocysts of at least one species of protozoa known to cause coccidiosis,said composition containing 0.8% by weight of alkali metal dichromate;and instructions for administration of said composition to an animal.

[0106] The preferred methods for isolation, concentration, flotation,sporulation, monitoring, separation, and sterilization, are nowdescribed.

[0107] Oocyst Isolation

[0108] The initial isolation of oocysts from the gross fecal matter isdesired to remove large debris and fecal matter from a manure slurry.Thus, the step begins with a manure slurry highly contaminated withgross particles and results in a aqueous manure slurry substantiallyfree of gross particles. Although isolation may be achieved by one ofthe above methods known in the art, isolation in the present inventionis accomplished by filtering. In a preferred embodiment, the filtrationis by sieving.

[0109] In one embodiment, the initial isolation is achieved bycollecting manure from host animals, mixing the manure with domesticwater, and then sieving. In one embodiment, the process begins withcollected manure, e.g., a batch of several hundred pounds, made into aaqueous slurry and processed to concentrate oocysts as a suspension in arelatively small volume of aqueous medium, e.g., a several hundred boundbatch of manure may ultimately yield about two liters of oocysts in anaqueous suspension (see FIG. 5A, “Challenge Suite”, steps 1-3B). In thisembodiment of the invention, sieving is by means of shaker screens, suchas multiple tier shaker screens.

[0110] In one embodiment, the manure from the chickens is placed into amixing vessel either by hand, e.g. using a shovel, or by using amechanical dumper. Domestic water is added at a minimum ratio of about 1gallon per each six birds' manure (see FIG. 5A, steps 3A and 4).Alternatively, the collected manure is mixed with domestic water at aratio from about 2 to about 6 pounds of collected manure per gallon ofwater. As manure quantity is approximate and dilution is realized byusing an approximation of the manure quantity, the dilution range istherefore also approximate. The slurry is then mixed until homogeneous.Feathers and other large, floating debris may be skimmed off the surfacewith an appropriate tool, e.g., a wire screen.

[0111] Typically, a sample is taken from the homogenous slurry prior tothe screening/isolation process to assess oocyst count. Such count isdone, for example, by microscopic visual examination. The homogenousslurry is then pumped onto a two-tier vibratory shaker screen. The topscreen can be from about 150-mesh to about 350-mesh while the bottomscreen can be from about 25-mesh to about 75-mesh. In a preferredembodiment, the top deck is equipped with a 50-mesh screen, the “topscreen,” while the lower deck has a 250-mesh screen, the “bottomscreen.” Larger unwanted fecal solids are separated at the top deck50-mesh screen while smaller unwanted fecal solids are separated fromthe slurry at the lower deck, 250 mesh screen. A preferred flow rateonto the top deck screen is approximately 1 liter per minute per 12.1m². The optimal flow rate of the pumping varies with the solids contentand the condition of the screen. Larger or smaller screens may be useddepending on the scale of the operation.

[0112] The oocysts are contained in the liquid fraction of thescreening/isolation process. If the solid material coming off either thetop or the bottom screen is too wet, recovery is unacceptably low asisolation of the oocysts from the homogenous slurry is not occurring. Onthe other hand, if too much water is removed, the solids stick to thescreen and do not clear themselves. Eventually, depending on solidmatter content and flow rates, so much material can accumulate that itmay be necessary to remove it. The screens can be lubricated with water,allowing the screens to clear themselves. The liquid fraction iscollected and sent on for concentration while the solid fraction isdiscarded (see FIG. 5A, step 6).

[0113] The solids coming off both of the screens are checked for oocystsand then discarded. If less than 5% of the oocysts loaded onto thescreens are found in the solids, then the solids are discarded. If morethan 5% of the oocysts loaded onto the screens are found in the solids,the solids are resuspended in an aqueous slurry and recycled through thesieves. The liquid that passes through both screens, the filtrate, isthe fraction that contains the oocysts. This filtrate is collected intoa receiving vessel (see FIG. 5A, step 7) and then sent to a centrifuge,preferably a bottle centrifuge or, alternatively, a decanter centrifuge.

[0114] In this non-limiting embodiment, the sieving method can becarried out in temperatures ranging from a low temperature thatsubstantially avoids freezing to a high temperature that substantiallyavoids damage to the oocysts, preferably at room temperature. Lowertemperatures, about 4° C., are preferred when sieving procedures takeover more than three hours to protect the viability of the oocysts. Inaddition, the sieving process can be carried out at any rate throughputallows as long as the screens do not accumulate excessive solid matterand at a rate rapid enough to prevent the manure from drying. As withother steps of the invention, equipment should be clean prior to use.

[0115] Concentrating the Filtrate

[0116] The liquid oocyst-containing fraction recovered, from any one ofthe preceding methods used to initially isolate oocysts, is thenconcentrated. Isolated oocysts are concentrated to increase spqrulationrates and output. Concentration is realized by various means, includingsubstantial separation from the aqueous slurry, centrifugal-basedseparation or centrifugal-based separation followed by filtration.Concentrating the oocysts is accomplished by utilizing one or more ofsuch techniques in combination with others. As used herein,centrifugal-based separation includes processing in either a mechanicalrotated centrifugation or a static hydrocyclone. In one embodiment,centrifugation is by decanter centrifugation. Other methods are alsoknown in the art and disclosed herein. Centrifuge scale and capacityvaries by batch size. For larger batch size, the use of a decantercentrifugation or use of a hydrocyclone would be preferred. For smallerbatch size, bottle centrifugation is preferred.

[0117] In one method, a combination of first sieving the collectedmanure, as described in the preferred sieving process above, is followedby continuous centrifugation and then filtration. In this methodpreliminary purification is achieved by sieving a homogenous slurry ofcollected manure through sieves having progressively smaller openings.Further purification is achieved by continuous centrifugation of theliquid fraction captured from the sieving process using a suspensionwherein the suspension has a specific gravity preferably between about1.01 to about 1.08 g/l. As a further purification step, the solidmaterial recovered from the centrifugation, which contains the oocysts,is re-slurried and filtered using a membrane of a pore size that retainsthe oocysts, but allows the passage of smaller material, includingbacteria. This process eliminates the flotation step, as describedbelow.

[0118] In an example of a further alternative procedure, the materialpassing through the sieves can be further purified by being collectedand pumped into a continuous flow centrifuge maintained at about 40-50°F., discarding the centrate, and collecting the solid materialcontaining the oocysts (such as seen in FIG. 5A, step 8).

[0119] In a preferred embodiment, the filtrate recovered from themulti-screen screening/isolation process described above is thenconcentrated using a centrifuge. In one embodiment, the filtraterecovered from the multi-screen screening process described above isthen concentrated using a bottle centrifuge. In a preferred embodiment,the filtrate recovered from the isolation/screening process is thenconcentrated using a bottle centrifuge wherein the filtrate is pouredinto centrifuge bottles and centrifuged at 1200×g for about ten minutes.The solid fraction formed contains the oocysts. The supernatant is thenpoured off (see FIG. 5A, step 8) and, if the solid fraction volumeallows, more filtrate is poured on top of the solid fraction. At somepoint, the solid fraction will need to be removed as more oocystscollect. The oocysts may be loosened or removed with a spoon or spatula.Residual material that is still in contact with the centrifuge bottlesmay be rinsed out using a minimal amount of domestic water. The oocyststhen require suspension in sufficient water to bring the solids contentto less than about 70% of the total volume of the suspension, preferablyless than about 60% of the total volume, and most preferably less thanabout 50% of the total volume, and should bring the suspension tosubstantial homogeneity. During this suspension process mixing of theaqueous suspension should be sufficient to keep the solids suspended,but the mixing should not create foaming. This suspension may then befurther processed by the flotation step, described herein below.

[0120] In an alternative preferred embodiment, the filtrate from themulti-screen screening process described above, is pumped into adecanter solid bowl continuous centrifuge at a rate of approximately 3to 4 liters/min. The decanter is set with a bowl speed of at least about4000 RPM and a conveyor speed of at least about 2500 RPM and no morethan about 3600 RPM. A receiving vessel is placed to catch the solids asthey are expelled from the solid discharge of the decanter centrifuge.Under these conditions, solids are discharged as a runny paste. Theliquid coming out of the liquid discharge of the decanter centrifuge ischecked for oocysts and discarded if the liquid contains less than about2% of oocysts initially loaded into the decanter centrifuge. If theliquid waste contains greater than or about 2% of oocysts initiallyloaded into the decanter centrifuge, the liquid should be re-mixed withthe solids and run again until the liquid waste contains less than 2% ofoocysts initially loaded into the decanter centrifuge.

[0121] In a preferred embodiment, once all the filtrate is pumped intothe decanter centrifuge, the centrifuge is allowed to run for a periodof time sufficient to move the residual solids out of the decanter. Inone embodiment, this period of time is more than about two but less thanabout five minutes. Use of various sized centrifuges will vary theperiod of time and may be adjusted by one skilled in the art. In apreferred embodiment, the speed of the bowl is then lowered to about1000 RPM and the speed of the conveyor lowered to about 500 RPM. Thelength of time and the bowl speed also varies according to batch sizeand can be properly adjusted by one skilled in the art. Domestic watermay be sprayed into the access port to wash solids off the innersurfaces of the body of the decanter. The solids are then moved to anappropriate volume centrifuge bottle for the flotation step, describedbelow. It is important to clean the equipment after each run and may beaccomplished by the use domestic water sprayed from a hose in order toobtain greater yield.

[0122] In yet another embodiment, a hydrocyclone is used to concentratethe filtrate obtained from sieving. It has been discovered that ahydrocyclone, traditionally used in the petrochemical and environmentalscience fields is useful for concentrating oocysts. Hydrocyclones usethe principle of centrifugal separation to remove or classify solidparticles from a fluid, based on size, shape, and density. The use of ahydrocyclone, not known to be used for living organisms, was previouslybelieved to fatally damage the oocysts due to intense sheer forces. Theinstant invention provides a method of utilizing a hydrocyclone toconcentrate oocysts. In one embodiment, the hydrocylone used is aDorr-Oliver DOXIE Type 5 Hydrocyclone (available from GL&V/Dorr-Oliver,Millford, Conn.).

[0123] In a preferred embodiment involving the use of a hydrocyclone, areservoir containing the filtrate obtained from sieving is connected toa pump. The pump delivers the filtrate to the hydrocyclone at a pressureof between about 120 psi and about 130 psi and at a feed rate from about1 to about 3 gallons per minute, preferably about 2 gallons per minute.A preferred hydrocyclone has one inlet and two outlets. Each outlet isequipped with a needle valve to regulate the flow through each orifice.By regulating the flow between the upper and lower outlets, it ispossible to remove a significant amount of liquid through the upperoutlet while retaining most of the denser materials, including theoocysts, in a concentrated suspension through the lower outlet. In apreferred embodiment, a 2 to 1 ratio between the flow of materialcollected from upper outlet and lower outlet. Such 2 to 1 rationproduces an optimal recovery of oocysts. The recovered concentratedmaterial, that is, the material collected from the lower outlet, may berecycled through the hydrocyclone for greater concentration if furthervolume reduction is desired. The suspension collected from the upperoutlet is discarded. For large volumes of filtrate, it may beadvantageous to operate hydrocyclones in parallel or utilize largerscale equipment to increase throughput.

[0124] Floating the Oocysts

[0125] To further isolate the oocysts collected from the concentrationmethods described above from unwanted solids, such as fecal matter,grit, etc., the oocysts are floated to the top of a solution usingdensity variations. In an alternative embodiment, the oocysts may beadded to a sucrose solution and centrifuged. In yet another alternativeembodiment, the oocysts may be added to a sucrose solution and thenfiltered. In a preferred embodiment, the oocysts are floated to the topof a solution comprising domestic water and high fructose corn syrup andhaving sufficient density to allow the oocysts to float to the top ofthe suspension while the heavier unwanted solids migrate to the bottomof a holding vessel or vessels. In a preferred embodiment, the oocystsare isolated from the dense solutions using centrifugation. The oocystsare then recovered from the liquid phase in this step of the invention.

[0126] In one embodiment, the solid material containing the oocystsrecovered from centrifugation is transferred to a mix tank and to aconcentrated sucrose or high fructose corn syrup (HFCS) of volume equalto that of the oocysts is added. A water of a volume equal to that ofthe oocyst/HFCS solution is added for a total final volume of about fourtimes the volume of the initial solids (FIG. 5A, steps 9-11). The finalmixture is then pumped into a continuous centrifuge at a rate to allowthe oocysts to remain in the centrate and solids are discarded (FIG. 5B,steps 12-13). If desired, further concentration of the oocysts anddilution of and substantial removal of the residual sugar solution isaccomplished by addition of domestic water and continuous flowcentrifugation at a feed rate which allows separation of the phasecontaining the oocysts from the sugar solution phase. In an alternativeembodiment, the oocysts/HFCS solution can be centrifuged in a bottlecentrifuge. In this case, the supernatant is discarded and the oocystsin the resulting solid fraction are resuspended in water. In yet anotheralternative embodiment, the residual sugar can be removed by filtrationusing filters with a pore size which excludes the oocysts. Whenfiltration is used, tangential flow is preferred. Tangential flowfiltration is characterized in that an influent stream is separated intotwo effluent streams, known as permeate and retentate. The permeate isthat fraction which has passed through the “semi-permeable” membrane (orfilter pad). The retentate is that stream which has been enriched withthe solutes of suspended solids which have not passed through themembrane (or filter pads). Water can be continually added to theretentate vessel at the same rate at which the sucrose-rich permeate isleaving in order to avoid over concentration of the solids. Oncesufficiently filtered, the retentate, containing the isolated oocysts,can then be stored in any suitable medium and temperature untilsporulation. In one embodiment, the isolated oocysts are placed insterile water and stored at about 2-8° C.

[0127] In a preferred embodiment, the decanter centrifuge method ofconcentration, described above, is used to concentration the filtrateretained from sieving and the volume of solids obtained from a decantercentrifuge is measured by volumetric measurement. Such measurement maybe taken by centrifuging about 50 ml of the concentrated filtrate forabout 10 minutes at 1,500×g (r_(average)) in a centrifuge with 50 mlconical tube adapters. Any centrifuge that produces the preferred forceson the filtrate may be used. The percent solids is calculated bymultiplying the volume of the solid by 2. Other well known methods mayalso be used to calculate solids and can be determined by one skilled inthe art. The solids content is then adjusted to less than 60% solids,with domestic water, if necessary. More preferably, the percent solidsis brought to below about 50% solids by the addition of domestic water,and most preferably the percent solids is brought to below about 40%solids by the addition of domestic water.

[0128] Then a HFCS solution, in a percent volume from about 30% to about40% of the solid collected from the concentration/centrifugation step,is added. This typically brings the density of the liquid phase up tothe point where the oocysts float. The density of the liquid is broughtup to at least 1.09 g/ml and can be brought up to an amount higher than1.09 g/ml. The density of the liquid is preferably between 1.09 g/ml andabout 1.20 g/ml, more preferably to be between 1.09 g/ml and about 1.14g/ml, and most preferably to be between about 1.09 g/ml and about 1.10g/ml. If the density of the liquid is less than 1.09 g/ml, remix theoocyst-containing liquid with additional HFCS solution until the densityis at least 1.09 g/ml. This dense liquid is then poured into vesselsproper for centrifuging, the vessels are balanced with respect oneanother in their placement in the centrifuge, and then centrifuged.

[0129] In a preferred embodiment, the dense liquid is centrifuged at atemperature from about 4□ C to about 10□ C. The density of the liquidphase is then measured following the first centrifuge run using methodswell known in the art. If the density of the liquid is less than 1.09g/ml, one should re-mix the liquid phase and the solid phase and addmore high fructose corn syrup solution to obtain a density of 1.09 g/mlor greater. These steps can be repeated if necessary in order to obtainthe highest yield of oocysts.

[0130] In a preferred embodiment, to the resuspended oocysts from theconcentration step described above, is added a volume of HFCS equal toabout 30% to about 40% of the volume of the solid fraction. HFCS isadded until the density of the liquid phase is brought up to the pointwhere the oocysts float, a density of about 1.09 to about 1.14 g/ml. Theentire suspension of oocysts in the HFCS/domestic water suspension isthen separated from the HFCS (see FIG. 5B, step 12).

[0131] In one embodiment, the HFCS/domestic water suspension is pouredinto centrifuge bottles, balanced with respect one another in theirplacement in the centrifuge, and centrifuged for about 15 minutes at3750×g (rmax) and at a temperature from about 4° C. to about 10° C. Thebuoyant oocysts float to the top of the suspension while heavierunwanted solids settle to the bottom of the bottles. The solidscontained in the supernatant should contain no more than about 40%solids. If the percent solids found in the supernatant, measuredaccording to the volumetric method described above, is higher than about40%, then the density is too high and the entire suspension needs to bediluted with domestic water and re-centrifuged.

[0132] In using the bottle centrifuge method of centrifuging, a largenumber of the oocysts will remain in contact with the bottle near thetop of the supernatant. This oocyst-containing material may be freed andreturned to the supernatant, for e.g., by swirling the bottles or byusing a tool, such as a spatula. This will not disturb the solid phase.The bottles may be swirled by hand at room temperature to remove thecrust of oocysts on the bottle. In larger batch sizes the vessel usedfor centrifuging can be cleaned by those methods familiar to one skilledin the art in order to clean the vessel and recover a higher percentageof oocysts.

[0133] The supernatant is then poured off into a vessel. If using thebottle centrifuge, rotating the bottles while pouring helps rinse theoocysts off the sides. The solid fraction can then be discarded. Thesame centrifuge bottles can then be refilled and the process repeateduntil all of the dense liquid has been centrifuged. The oocysts are nowready to go to the second concentration step which removes residualsucrose.

[0134] Concentrating the Oocysts after Flotation

[0135] The liquid fraction from the floatation step described above isthen diluted with domestic water and the separated from the HFCS. Inthis process, the oocysts are concentrated prior to the sporulationstep. The concentrated oocysts are then diluted yet again and held priorto sporulation.

[0136] In one embodiment the liquid phase recovered from the flotationcentrifugation step is first diluted with domestic water till theoocysts sink and then centrifuged to capture the oocysts in the solidphase (FIG. 5B, steps 15-18) to remove a substantial amount of HFCS. Inanother embodiment, the liquid phase recovered from the flotationcentrifugation step is first diluted with domestic water till theoocysts sink and the suspension is processed with a hydrocyclone. Inusing the hydrocyclone, the upper fraction is recovered. Subsequentseparation of the HFCS, the concentrated oocyst containing suspension isagain diluted with domestic water and transferred to a holding vesselprior to sporulation (FIG. 5B, steps 19-20).

[0137] In a preferred embodiment, the volume of the liquid fractionrecovered from the flotation step is measured and a sample is taken toassess oocyst count. Sufficient domestic water is added to lower thedensity of the supernatant to less than about 1.04 g/ml. This allows theoocysts to sink. The density is measured following the addition of thedomestic water using techniques well know in the arts. If the density isnot less than about 1.04 g/ml and/or the oocysts have not sunk,additional domestic water is added until such density is reached and/orthe oocysts sink. The oocyst suspension is then poured into centrifugebottles and centrifuged for about 10 minutes at 1200×g from about 4° C.to about 10□ C. The supernatant is tested for oocyst presence bycounting using a microscope and hemocytometer and the supernatant isdiscarded if an acceptable amount of oocysts are counted in thesupernatant. An acceptable amount of oocysts in the supernatant is fromabout 1% to about 5%, preferably about 2%, of the total oocysts loadedat the beginning of flotation step. More of the mixture from theflotation step is then poured on top of the solid fraction generated bycentrifugation. While not necessarily being resuspended, the solidfraction is loosened somewhat, particularly by inverting the bottle afew times. The resuspended solid fraction suspension is then centrifugedas before, for about 10 minutes at 1200×g from about 4□ C. to about 10□C., and the process is repeated until the flotation step mixture has allbeen centrifuged.

[0138] When using the bottle centrifugation method, at this point, thereshould be several bottles, each with a solid fraction in the bottom.Note, however, with larger batch size the vessel or vessels vary withequipment that is of appropriate volume and recovery methods may bedetermined by one skilled in the art. The solid fractions in thecentrifugation vessels are then resuspended by shaking them with aminimal amount of domestic water. The solid fractions are rinsed intoone or two of the bottles and the bottles filled and balanced with waterif necessary. These bottles are centrifuged one last time as before, forabout 10 minutes at 1200×g from about 4° C. to about 10□ C. Thesupernatant is then discarded. Any loose solid fractions that comes outwith the supernatant can be ignored. The solid fraction is thenresuspended in a minimal amount of domestic water and stored in a singlebottle from about 2° C. to about 5□ C. pending sporulation whilefreezing should be avoided.

[0139] In an alternative embodiment, the HFCS in the liquid phaserecovered from the flotation step can be remove by filtration usingfilters with a pore size which excludes the oocysts. When filtration isused, tangential flow is preferred. Tangential flow filtration (TFF) ischaracterized in that an influent stream is separated into two effluentstreams, known as permeate and retentate. The permeate is that fractionwhich has passed through the “semi-permeable” membrane (or filter pad).The retentate is that stream which has been enriched with the suspendedsolids which have not passed through the membrane (or filter pads). Oncesufficiently filtered, the retentate, containing the isolated oocysts,can then be stored in any suitable medium and temperature untilsporulation. In one embodiment, the isolated oocysts are placed insterile water and stored at about 2-8° C. Note that the tangential flowfiltration is an alternative embodiment to concentrating the oocystsafter flotation and TFF at this step should not be confused with TFFused during sterilization.

[0140] In an alternative embodiment, the volume of the liquid fractionrecovered from the flotation step is measured and a sample is taken toassess oocyst count. Sufficient domestic water is added to lower thedensity of the supernatant to less than about 1.04 g/ml. This allows theoocysts to sink. The density is measured following the addition of thedomestic water using techniques well know in the arts. If the density isnot less than about 1.04 g/ml and/or the oocysts have not sunk,additional domestic water is added until such density is reached and/orthe oocysts sink. The oocysts suspension is then processed through ahydrocyclone at a flow rate of about 2 gallons per minute and at apressure between about 120 psig and about 130 psig.

[0141] Batch size and scale will lead one skilled in the art to utilizevarious centrifugation processes based on batch size while one skilledin the art will also adjust the correct centrifugation speeds based onthe batch size.

[0142] Sporulation

[0143] Sporulation is performed to transform the cleaned andconcentrated oocysts into their next life form, the sporulated oocyst(see FIG. 5B, “Sporulation Suite”). Sporulation may be performed in anysuitable container, however, a fermentation vessel is preferred in orderto best control temperature, dissolved oxygen, pH, and mixing inaddition to monitoring these parameters of the sporulation medium. Thecapacity of the sporulation vessel varies with batch size and can beadequately selected by one skilled in the art. A preferred fermentor isthe New Brunswick BioFlow 3000 (available from New Brunswick ScientificCompany, Edison, N.J.).

[0144] Sporulation is achieved by subjecting the oocysts to an oxidativechallenge. In this step, the oocysts are contacted with an oxidizingagent which is effective to promote sporulation but does not result inthe death of the oocysts. As described below, the oxidizing agentcomprises a principal oxidant other than a source of dichromate.Preferably, the sporulation medium is substantially devoid of potassiumdichromate, an alkali metal dichromate, dichromate ions or otherdichromate salt. In a preferred embodiment, the sporulation mediumcontains less than about 0.8% by weight of alkali metal dichromate, orless than about 0.6% by weight of alkali metal dichromate, or less thanabout 0.4% by weight of alkali metal dichromate, or less than 0.2% byweight of alkali metal dichromate, more preferably 0.1% by weight ofalkali metal dichromate, and most preferably the sporulation medium issubstantially free of an alkali metal dichromate. In another preferredembodiment, the sporulation medium contains less than about 3.0% byweight of dichromate ions. In yet another preferred embodiment, thesporulation medium contains less than about 1.5% by weight hexavalentchromium.

[0145] In one embodiment of the invention, oocysts concentrated by themethods and processes of the instant invention and described above arecollected over a time period sufficient to create a batch size suitablefor a fermentation vessel of desired volume. The collected concentratedoocysts are then deposited in to a sporulation vessel, an oxidizingagent is added and sporulation is allowed to occur (see FIG. 5B, step21). Domestic cold water is used to rinse the container or containersholding the oocysts prior to their contact with the fermentation vessel.The sporulated oocysts and the rinse is then transferred to a separationdevice (FIG. 5B, step 22). The instant invention also provides forsporulation by depositing concentrated oocysts in a fermentation vessel,subjecting the oocysts to an oxidative challenge by contacting theoocysts with an oxidizing agent, such as oxygen or sodium hypochlorite,in an aqueous medium, wherein the percent saturation of dissolved oxygenin the medium is maintained at preferred levels, pH is controlledbetween preferred levels by the alternative addition of an acid or abase, the suspension is mixed to near homogeneity, and temperature isbetween preferred temperatures over a preferred period of time. In afurther aspect of the instant invention an anti-foaming agent is addedduring the sporulation process. Preferably solids do not exceed morethan about 50%. Preferably solids are less than about 35%. Morepreferably still, solids are less than about 25%. Sufficient domesticwater is added to the sporulation vessel to achieve this ratio betweensolid and liquid phase. The liquid phase in the fermentation vessel istermed the sporulation medium.

[0146] In a preferred embodiment the oxidizing agent used is oxygen.Oxygen may be added in the form of air or as pure oxygen.

[0147] In an alternative embodiment, sufficient 5.25% sodiumhypochlorite is added to the fermentor to achieve the following initialconcentrations upon dilution of active chlorine concentrations for theindividual subspecies. The values are approximate and indicate preferredmaximum concentrations that do not inhibit sporulation: TABLE 1 Spp. ofEimeria weight % E. Acervulina 0.01 E. Maxima 0.05 E. Tenella 0.05

[0148] During sporulation, the percent saturation of dissolved oxygencontent in the aqueous medium is maintained at at least 30% ofsaturation, preferably at at least 40% of saturation, and morepreferably at least 50% of saturation. Percent saturation of dissolveoxygen is controlled, by supplying air or molecular oxygen, to achieveconsistent and higher yields of sporulated oocysts.

[0149] In a preferred embodiment of the invention, percent saturation ofdissolved oxygen is maintained by bubbling air through the mixture at arate sufficient to meet the above ranges. Pure oxygen may also bebubbled through the mixture to maintain the requisite percent dissolvedoxygen. Care should be taken so that the flow of oxygen is not so rapidas to cause foaming. If desired, an anti-foaming agent may be added,such as Antifoam A (available from Sigma-Aldrich, St. Louis, Mo.).Oxygen is added by any means practicable. Oxygen may be added by addingboth air when lesser flow rates are needed. e.g., when oxygenconsumption is relatively low to peak sporulation, to maintained thepreferred percent dissolved oxygen saturation while molecular oxygen maybe added when the need is greater, e.g., when oxygen consumption isgreatest. Oxygen is preferably added at a flow rate of from about 0.1 toabout 2.0 liters of gas per liter of material and more preferably fromabout 0.3 to about 0.5 liters of gas per liter of material. The flowrate may be kept constant despite a greater need to maintain preferredpercent saturation of dissolved oxygen as the gas added may consist ofair when less oxygen is needed and may consist of molecular oxygen whenmore oxygen is needed. The preferred fermentor automatically convertsfrom the addition of air to molecular oxygen as needed while controllinga nearly constant flow rate.

[0150] The pH level is preferably maintained from about 7.0 to about7.7, more preferably from 7.2 to about 7.5, and more preferably stillthe pH is maintained about 7.4. The pH level of the sporulation mediumis controlled by adding an acid or a base. In a preferred embodiment,either sodium hydroxide (5N) or sulfuric acid (5N) is alternativelyadded to the sporulation medium as needed to maintain the pH near 7.4.When using a fermentation vessel, the acid and/or the base may be addedby using a fermentation vessel's automatically controlled peristalticpumps on the fermentor.

[0151] The temperature of the sporulation medium is controlledthroughout sporulation. Oocysts are placed in a sporulation vessel at atemperature from a temperature that substantially avoids freezing toabout 43° C.; preferably between about 15° C. to about 38° C.; and morepreferably between about 20° C. to 30° C. and more preferably still atabout 28° C.±1° C. It will be apparent to those of ordinary skill in theart that the rate of sporulation is temperature dependent, so that thetime required for sporulation will generally be less at highertemperatures.

[0152] Throughout the sporulation process, the sporulation medium ismixed. Any suitable method of mixing can be used to mix the sporulationmedium to about a homogenous state. The exact method of mixing variesdepending on the container used. For example, when bottles or flasks areused, mixing can be achieved by the use of shakers, or magnetic ormechanical stirrers. When vats or fermentors are used, a mechanicalstirrer, such as a paddle stirrer is preferred.

[0153] Although sporulation is substantially complete within 12 to 18hours, removal of the sporulated oocysts prior to about 72 hoursdecreases viability. Therefore, sporulated oocysts are preferably keptunder the above sporulation conditions for a preferred time period toprovide a more stable population of sporulated oocysts. The oocysts arepreferably maintained in the above conditions for approximately 72 to120 hours, more preferably for 72 to 110 hours, and more preferablystill for 72 to 96 hours, to allow sporulation to occur.

[0154] Sporulation start point, end point and rate may be monitored bymonitoring: (1) the rate at which oxygen must be added to thesporulation medium to control percent saturated dissolved oxygen; and/or(2) by monitoring the amount of acid or based required to be added tocontrol the pH of the sporulation medium. It has been discovered thatsporulation results in an increase in oxygen consumption, as evidencedby a decrease in dissolved oxygen in the sporulation medium, and anincrease in pH, that is, if percent saturation of dissolved oxygen andpH are not controlled. When no additional oxygen is added to thesporulation medium, sporulation is indicated by a drop in dissolvedoxygen to less than 60% of saturation, more preferably less than 40%,and more preferably still less than 20%. The change in dissolved oxygencan also be measure in terms of percent change. Thus, sporulation canalso be indicated by a decrease of at least 10% (i.e., from 50% to 40%),preferably at least 20%, more preferably at least 30%, and morepreferably still at least 40% in dissolved oxygen content as expressedin percent of saturation (see FIG. 1A). When pH is not controlled by thealternative addition of an acid or a base, increase in pH of at leastabout 0.25 pH units, more preferably at least about 0.5 pH units, isindicative that sporulation is occurring (see FIG. 2).

[0155] The change in dissolved oxygen and pH do not occur independently.An increase in the oxygen consumption indicates the start point ofsporulation. Note, however, that background oxygen consumption will beseen as the sporulation medium is not sterile at this point and sovarious bacteria will be consuming oxygen as well as the oocysts.However, the increase in oxygen consumption will be significant over thebackground oxygen consumption so that the sporulation start point, endpoint, and rate, including peak, are readily ascertainable. A decreasein oxygen consumption indicates a drop in sporulation rate. Once oxygenconsumption becomes low and consistent, sporulation is substantiallycomplete, usually after about 18 hours. However, as mentioned above, thesporulated oocysts should be maintained under the sporulation conditionsfor at least an additional 36 to 48 hours to increase yield. Monitoringof sporulation will assist the practitioner in reaching higher yields ofviable sporulated oocysts. Optionally, sporulation can be confirmed bymicroscopic examination of the oocysts. However, the method of presentinvention obviates the need for sampling and microscopic examination.

[0156] Sterilization

[0157] Following sporulation, the sporulated oocysts, are removed fromthe sporulation vessel, and washed free of the sporulation medium andconcentrated by any suitable method, preferably filtration. The entiresterilization process is generally conducted in two phases: (1)contaminants are first removed non-aseptically (see FIG. 5C, steps23-25); followed by (2) disinfection of sporulated oocysts mediumcarried out under sterile conditions (see FIG. 5C, steps 26-28). Thepurpose of this process is to collect sporulated oocysts and filter outcontaminants. A further purpose is to concentrate oocysts, preferably byfiltration. However, centrifugation may also be used to concentrate thesporulated oocysts. A further purpose is to sterilize the suspensionwith a disinfectant, preferably sodium hypochlorite (leaving thesporulated oocysts intact), then to remove the disinfectant from andthen concentrate the sporulated oocysts. Then, to the sporulatedoocysts, is added an appropriate quantities of buffer and antibiotic,preferably PBS and gentamicin (FIG. 5C, steps 30-31). This sporulatedoocysts-containing suspension is then transferred into suitable storagecontainers for bulk storage prior to final packaging for distribution toconsumers.

[0158] In one embodiment, separation of the sporulated oocysts from thesporulation medium may be achieved by centrifugal-based separation, suchas by bottle centrifuge, decanter centrifuge, or by hydrocyclone. Thevolume of the batch size will be determinative of the mode ofcentrifugal-based separation and can be determined by one skilled in theart. The solid fraction from any one of the centrifugal-based separationmethods is recovered. If more than about 5% of the oocysts loaded intothe centrifugal-based separation unit are in the refuse fraction, aliquid fraction in this embodiment, said fraction is mixed with thesolid fraction and recycled through the centrifugal-based separationunit. The recovered solids are then diluted to a volume appropriate forsterilization, preferably by filtration, more preferably by tangentialflow filtration.

[0159] In a preferred embodiment, once sporulation is complete, theresultant aqueous suspension of sporulated oocysts is transferred fromthe fermentation vessel into a receiving vessel of appropriate volume.The transfer of the oocysts from the fermentation vessel is preferablyaccomplished by using air forced through the fermentation vessel, e.g.,pressurizing the headspace, thereby forcing the sporulated oocysts intothe awaiting container. A sample of the sporulation medium from thecontainer is then taken to assess sporulated oocyst count andsporulation ratio. Any material still in contact with the fermentationvessel may be removed by using a rinse, e.g., a sufficient amount ofdomestic water, and the rinse may then be combined with the suspensionof sporulated oocyst already transferred from the fermentation container(FIGS. 5B-C, step 22). Again, a sample is then taken to assesssporulated oocyst count and sporulation ratio in an effort to ascertainyield. The manner and method in which the sporulated oocysts areharvested from the fermentation vessel varies with the batch size andtype of fermentation vessel and may be properly determined by oneskilled in the art.

[0160] After removal of the suspension of sporulated oocysts from thefermentation vessel, the oocysts are allowed to settle from sporulationmedium over a period of several hours, e.g., 8 to 20 hours, while themedium and the oocysts are held at a temperature from a temperature thatprevents freezing to about 10° C., more preferably from about 2° C. toabout 6° C., and most preferably about 4° C. Sporulated oocysts settleto the bottom of the storage container while contaminants remainsuspended or dissolved in the aqueous layer. In a preferred embodiment,the supernatant is decanted, poured or pumped off using a smallperistaltic pump or other method suitable to the volume being removed.Domestic water is then added at a sufficient volume to resuspend thesporulated oocysts. Settling can be carried out in the receiver or in aseparate settling vessel.

[0161] The sporulated oocysts, now resuspended after collection fromsettling, are then separated from the sporulation medium. In a preferredembodiment, separation is by filtration. However, any appropriate meansmay be used to separate the sporulated oocysts from the sporulationmedia. In a more preferred embodiment, the filtration process is bymeans of tangential flow filtration. Tangential flow filtration (“TFF”)is used in this procedure to separate sporulated oocysts from othermaterial that may be present in the suspension, e.g., grit, othermicroorganisms, etc. In addition to the filter membrane, two essentialparts of the TFF system are a retentate vessel, which holds thesporulated oocysts, and a low shear pump that circulates the retentatethrough the membranes and back into the retentate vessel. The oocystsare retained in the retentate while the permeate is discarded.

[0162] The pore size of the filter membrane should be small enough sothat sporulated oocysts cannot enter the pores, but large enough toallow bacteria to pass through. In one embodiment, the filter has a poresize of approximately 10-microns. In yet another embodiment the filterhas a pore size of approximately 5-microns. A preferred filtration unitis a Consep membrane unit manufactured by North Carolina SRT (availablefrom North Carolina SRT, Inc., 221 James Jackson Ave., Cary, N.C.27513). However, other filtration units may be used, such as thoseproduced by Millipore (available from Millipore Corporation, 80 AshbyRoad, Bedford, Mass. 01730). A preferred filter is the Spectra/Meshpolyester filters (Spectrum Laboratories, Inc., Rancho Dominguez,Calif.; cat no: 146524). Tangential filtration units such as an OPTISEPCL, OPTISEP, or CONSEP may be used, also available from North CarolinaSRT. Throughput can be increased by utilizing a larger scale filtrationunit. One skilled in the art will recognize that the type of filtrationunit needed depends on the volume of the sporulated oocyst suspension.In one embodiment, an OPTISEP CL unit is used to run about a 1 Lsporulated oocyst suspension. In another embodiment, a CONSEP unit isused to run about a 10 L sporulated oocyst suspension.

[0163] When a filtration process is applied to a sporulatedoocyst-containing medium the permeate is discarded. Water is added aspermeate is removed if the filtration process is conducted at a constantvolume. Filtration can be accomplished by gravity flow or by the use ofa pump, for example, a peristaltic pump. In a preferred embodiment, themixture is pumped tangentially over the filter. If a pump is used, therate of pumping varies with such well known factors as the surface areaof the filter, the path length, the total area of the flow channel, andthe pore size. Optimum pumping rates can be determined by one ofordinary skill in the art without undue experimentation as such flowrates are a function of surface area of the filtration unit and solidscontent.

[0164] In a further embodiment, the inlet and the outlet tubing for thetangential flow unit are placed into a vessel containing the sporulatedoocysts while the permeate tubing is placed in a separate vessel. Thepump, for example, a diaphragm pump, is then started to beginfiltration. A preferred flow rate is about 1 LPM per 160 cm². The pumprate may also be expressed in terms of lineal velocity. Linealvelocities may be between 20 and 50 centimeters per second. A preferredlineal velocity when using a CONSEP filtration unit is 28 centimetersper second. The pump may be kept running to maintain the flow ratethroughout the process. The permeate is sampled and, using a glassslide, observed for sporulated oocysts. The optical density of thepermeate sample is also measured using a spectrophotometer at 600 nm(OD₆₀₀). Circulation of retentate over the filter medium is continued ifthe concentration of oocysts in the permeate has not increased to orexceed a maximum tolerable level. An acceptable concentration is fromabout not more than 5% of the total sporulated oocysts loaded into thefiltration unit. If the sporulated oocysts concentration measures to orabove the maximum acceptable level, filtration is stopped. The permeateis recycled and mixed with the retentate and filtration is resumed.Filtration is stopped when the measured OD₆₀₀ is about less than about0.5 at a lineal velocity of about 28 centimeters per second. However,filtration may be stopped when the measured OD₆₀₀ is about 0.6, again ata lineal velocity of about 28 centimeters per second. Once the desiredOD is reached, oocysts from the membranes and the tubing are transferredto the retentate vessel and any oocysts remaining in the membranes andtubing are then flushed with water into the retentate vessel.

[0165] The retentate, containing the concentrated oocysts, is thenplaced in a vessel under refrigeration at about 4° C. from about 15 toabout 24 hours. This allows the sporulated oocysts to settle in bottomsolid phase and a liquid phase will normally form above the solid phase.The liquid phase is then substantially removed after refrigeration toreduce unwanted volume, e.g., by decanting, pumping, or siphoning. In analternative embodiment, the retentate is processed immediately afterfiltration. However, it is preferred to let the retentate rest asdescribed above.

[0166] Once the sporulated oocysts have been concentrated by filtrationthey can be sterilized by means of a chemical disinfectant orsterilizing agent other than an alkali metal dichromate, solubledichromate moieties, dichromate ions, or potassium dichromate.Sterilization processes are conducted in sterile environments. In apreferred embodiment, sterilization is accomplished within thefiltration device used to concentrate the sporulated oocysts. In analternative embodiment the retentate containing the sporulated oocystscan be washed from the filter and sterilization is accomplished in avessel separate from the filtration device. Any filtration unit used tosterilize the sporulated oocysts should be sterilized prior to theaddition of the unsterilized sporulated oocysts. In one embodiment, thefiltration unit is sterilized by autoclaving. In an alternativeembodiment, the filtration unit is sterilized by passing steam atapproximately 250° C. through the system for at least about 30 minutesat approximately 20 psi. In yet another alternative embodiment, the unitis chemically sterilized by treating the system with 5% sodiumhypochlorite for at least about 10 minutes wherein the sodiumhypochlorite contains at least about 5% available chlorine by weight.

[0167] The agent used for sterilizing the sporulated oocysts preferablyis one which kills bacteria and viruses, but does not kill thesporulated oocysts. Preferably, the disinfectant used kills theinfectious bursal disease (IBDV), chick anemia (CAV) viruses, andrelated bacteria. As IBDV is known to be a robust virus, a sterilizationagent that kills IBDV will kill other, less robust microorganisms aswell. An agent that eliminates IBDV is considered to substantiallyeliminate microorganisms.

[0168] In a preferred embodiment, the disinfectant used is sodiumhypochlorite. The concentration of disinfectant used varies with theagent chosen to accomplish sterilization. In more preferred embodiment,sodium hypochlorite is used at a concentration preferably in the rangefrom about 1% to about 10%, and more preferably in the range of about 2%to about 5% wherein the percent represents the percent of availablechlorine by weight. The time during which the sporulated oocysts areexposed to the disinfectant varies depending upon factors such as theconcentration of the disinfectant and the volume of the batch ofsporulated oocysts. In one embodiment, the sporulated oocysts aretreated with approximately 5% sodium hypochlorite, wherein the percentrepresents the percent of available chlorine by weight, from about 2 toabout 20 minutes, more preferably from about 5 to about 18 minutes, andmost preferably for about 10 minutes.

[0169] Once the filtration unit is sterilized, the vessel holding thesporulated oocyst suspension is then removed from refrigeration. Theclear upper layer is removed by pumping, pouring, or suctioning off thesupernatant, leaving the bottom sporulated oocyst fraction. The latterfraction is then transferred to a retentate vessel of adequate volume.The previous vessel is then rinsed with domestic water. The domesticwater rinse is then added to holding vessel and stirred by adequatemeans. In one embodiment, stirring is accomplished by means of amagnetic stir bar while the retentate vessel is sitting on a magneticstirrer. In another embodiment, stirring is accomplished by meansappropriate for the volume of the retentate vessel, such as with apaddle.

[0170] Next, a volume of about 10% aqueous sodium hypochlorite solutionthat is approximately equal to the volume of suspension in the retentatevessel is added to the sporulated oocyst suspension. The sporulatedoocyst suspension containing sodium hypochlorite should have a solidscontent of preferably less than about 30%, more preferably to less thanabout 25%, and even more preferably to less than about 15%, and mostpreferably to less than 7.5%. A solids content of less than about 7.5%is preferred as the reduced solids content produces a higher assuranceof sterility. When solids are brought to about 15%, there isapproximately 5% sodium hypochlorite, wherein the percent represents thepercent of available chlorine by weight, in the suspension. In analternative embodiment, the solids concentration is adjusted to about15% prior to sodium hypochlorite addition. After this addition, thesolids are 7.5% and the sodium hypochlorite is at 5%. With eitherembodiment, the suspension is mixed thoroughly by adequate means and, inone embodiment, allowed to stand for preferably 5 minutes, morepreferably 8 minutes, and most preferably about 10 minutes. Standingtime will vary depending on batch size and volume and may be adjusted asneeded. However, standing may be avoided and filtration may occurimmediately after dilution.

[0171] The retentate vessel containing the sporulated oocysts suspensionis then connected to the filtration unit. Autoclaved or otherwisesterile water is used as a water source during the sterilizationprocess. The retentate pump is then activated while the permeate line ispinched or clamped closed. Once any air bubbles have been substantiallyeliminated from the membranes and tubing the permeate line may be openedand directed to a collection vessel outside a sterile environment.Permeate is then analyzed to verify that there is no significant loss ofoocysts via the permeate.

[0172] In an alternative to using filtration to remove the sodiumhypochlorite from the oocyst suspension, centrifugation may be utilized.

[0173] While the hypochlorite-rich permeate leaves the filtration unit,sterile water is added to replenish the volume. Filtration is continuedand water is added as needed to control the percent solids duringfiltration. A sample is then taken from the permeate to determine totalchlorine level. Chlorine level can be detected using CHEMetrics VacuetteKit, available from CHEMetrics, Inc., Route 28, Calverton, Va., 20138.The total level of chlorine should is reduced to less than about 1 ppmby. When the permeate contains less than about 1 ppm of the chlorine theretentate, which contains the oocysts, also contains less than about 1ppm. Once the desired level of chlorine is reached, filtration may becontinued without adding more water to reduce the overall volume of thesporulated oocysts suspension.

[0174] When the desired retentate volume is reached, the pump feedtubing is removed from the retentate vessel and placed into a vesselcontaining sterile water and filtration is continued. Once substantiallyall of the suspension has been flushed out of the tubing and the filterhousing, the pump is shut off and filtration is complete.

[0175] Next, if desired, a buffer, such as PBS, and a bactericide isadded to the sterilized sporulated oocyst suspension. In a preferredembodiment, 1×PBS containing 60 Ag/ml gentamicin is added in a 1:1 ratioto the disinfected sporulated oocyst suspension to result in asuspension of sporulated oocysts in 0.5×PBS with 30 μg/ml gentamicin.The suspension is then stored under refrigeration, preferably around 4°C. for future use. In an alternative embodiment, 1×PBS is added to thesporulated oocyst suspension while no bactericide is added to thesporulated oocyst suspension. Average yields for all three subspecies,E. tenella, E. acervulina, and E. maxima, is about 70% for disinfectionand final filtration.

[0176] For the purposes of the present invention, sporulated oocysts andcompositions containing sporulated oocysts are considered sterile ifsamples of liquids containing the oocysts do not have detectable amountsof live bacteria, IBD virus or CAV virus. Detection of live bacteria canbe accomplished by any method known in the art. For example, bacteriacan be detected by incubation on bacterial agar plates at 35-37° C. for18 to 24 hours. One preferred method of testing is set forth in 9 C.F.R.113.27(1999), hereby incorporated in its entirety by reference. Briefly,to test for bacterial contamination, a sample of the preparation of thepresent invention can be innoculated into soybean casein digest mediumand incubated at 30 to 35° C. for 14 days. To test for fungalcontamination, a sample of the preparation of the present invention canbe innoculated into soybean casein digest medium and incubated at 20 to25° C. for 14 days. After the incubation period, the vessels can beexamined macroscopically for microbial growth. If growth cannot bedetermined reliably by visual examination, the judgment can be confirmedby microscopic examination.

[0177] Detection of IBDV virus or CAV virus can be by any method knownin the art. A non-limiting example of IBDV and CAV detection is by themethods set forth in 9 C.F.R. 113.47 (1999), herein incorporated in itsentirety by reference. Briefly, to test for CAV, MSB-1 cells from theMaine Biological Laboratories, Waterville, Me. are used as the indicatorcell line for Chicken Anemia Virus. MSB-1 cells are a lymphoblastoidcell line from a Marek's disease lymphoma that show cytopathic effectwhen infected with Chicken Anemia Virus. Cells are maintained inOpti-MEM® (Life Technologies, Gaithersburg, Md.) or other suitable mediaat 41° C. for at least 24 days prior to testing. Cells are subcultured10-12 times during the maintenance period with all but the lastsubculture resulting in a monolayer of at least 75 cm². The lastsubculture is at least 6 cm².

[0178] Three groups of MSB-1 monolayers are used for each test, anegative control group, a positive control group, and a test group. Atthe start of the 24 day maintenance period, the positive control groupis inoculated with 10^(5.75) TCID₅₀/ml of Chicken Anemia Virus, Del Rosstrain originally obtained from the Center for Veterinary BiologicsLaboratory (Ames, Iowa) and the test group inoculated with the testpreparation. The negative control group is inoculated with a preparationknown to be free of Chicken Anemia Virus. The cells are then maintainedfor at least 24 days as described above.

[0179] Two days after the last subculture, the three groups ofmonolayers are fixed and treated with a specific chicken polyclonalChicken Anemia Virus antibody (Hy-Vac, Adel, Iowa). The monolayers arethen washed and treated with a fluorescein labeled goat anti chicken IgG(H&L, Jackson Immunoresearch, West Grove, Pa.) and examined for specificfluorescence. If the positive control shows specific fluorescence andthere is no difference in fluorescence between the test and negativecontrol groups, the preparation is considered free of Chicken AnemiaVirus.

[0180] Alternatively, the presence of Chicken Anemia Virus can bedetected by the polymerase chain reaction (PCR). Three days after thelast subculture, DNA is extracted from the three groups of monolayersusing well established procedures. See, for example, Ausubel et al.,Short Protocols in Molecular Biology, 2^(nd) Ed., John Wiley & Sons,1992; Sambrook et al., Molecular Cloning, A Laboratory Manual, 2^(nd)Ed., Cold Spring Harbor Laboratory Press, 1989; Davis, et al., BasicMethods in Molecular Biology, Elsevier, 1986. Briefly, cells are lysedby two cycles of freezing to −80° C. and thawing at 37° C. Cellulardebris is removed by centrifugation at about 3,500×g for 20 minutes. Thesupernatant is treated with DNase and RNase A to remove cellularcontaminants and the proteins and/or virus precipitated withpolyethylene glycol. The precipitate is treated with Proteinase K andextracted three times with phenol/chloroform/isoamyl alcohol. DNA isprecipitated with sodium acetate-ethanol and pelleted by centrifugationat 14,000×g for 15 minutes. The resulting pellet is resuspended indouble distilled water and stored at −20° C.

[0181] Conserved regions of the Chicken Anemia Virus viral genome areamplified using standard techniques (Innis et al., PCR Protocols,Academic Press, 1990). Information on the Chicken Anemia Virus viralgenome for designing suitable primers can be found on databases wellknown to those in the biomedical arts such as the databases availablethrough the website of the U.S. National Institutes of Health. PCRproducts are analyzed by agarose gel electrophoresis and ethidiumbromide staining. If the PCR amplification does not result in a bandcorresponding to the band found in the positive control, the preparationis considered free of Chicken Anemia Virus.

[0182] One preferred, but non-limiting method for detection of IBDV inthe preparation of the present invention is the same as for ChickenAnemia Virus with the following changes. A preferred cell line used forIBDV testing is a primary chick embryo fibroblast cell line. Afteraddition of a sample of the preparation or 108.25 TCID₅₀ of IBDVoriginally obtained from American Type Culture Collection (ATCC VR-2041strain D78), the monolayers are maintained in basal medium Eagle (BME)or other suitable media at 37° C. and 5% CO₂ for at least 14 days.Detection is preferably accomplished using an IBDV specific polyclonalchicken antiserum and a fluorescein labeled goat anti-chicken IgG(Jackson Immunoresearch, West Grove Pa.). If the positive control showsspecific fluorescence and there is no difference in fluorescence betweenthe test and negative control groups, the preparation is considered freeof IBDV.

[0183] Alternatively, the test for the IBDV contamination can beaccomplished using PCR as described for Chicken Anemia Virus.Information on the IBDV viral genome for designing suitable primers canbe found on databases well known to those in the biomedical arts such asthe databases available through the website of the U.S. NationalInstitutes of Health. If the PCR amplification does not result in a bandcorresponding to the band found in the positive control, the preparationis considered free of Infectious Bursal Disease Virus.

[0184] Post-challenge performance improvement compositions may also beadded to the sterilized sporulated oocyst suspension. A preferredpost-challenge performance improvement composition is Propionibacteriumacnes (P. acnes) and can be added to the sporulated oocyst suspensionprior to the time of filling vials for consumer use. The P. acnes may beobtained from independent manufactures in 1-liter glass bottlessuspended in PBS or water and having gentamicin at a cell densityequivalent to 10¹² cells/ml of the pre-autoclaved P. acnes count. The P.acnes suspension is preferably included in the final vaccine at a dryweight dose equivalent to 50 μg per bird.

[0185] Storage

[0186] The instant invention also provides for compositions to storesterile sporulated oocysts. The sporulated oocysts can be held insterile water or other suitable diluent (see FIG. 5C, “Storage Suite”).In a further embodiment, an oxidizing agent is added to the storagecomposition. In one embodiment, the suspension of sporulated oocysts istransferred to vials that are prepared in a kit containing thesuspension as a ready-to-administer vaccine.

[0187] Sterile sporulated oocysts are preferably stored at anytemperature between room temperature and a low temperature thatsubstantially avoids freezing. In a preferred embodiment, the sporulatedoocysts are stored at between about 1° C. and about 10° C., morepreferably between about 2° C. to about 7° C., and in a most preferredembodiment between about 4° C. to about 5° C. In an alternativeembodiment, the sporulated oocysts are stored at a temperature between20° C. to about 30° C., more preferably between 22° C. to about 27° C.,and most preferably at about 25° C.

[0188] Although not necessary, a buffering agent may be added to thediluent in which the sporulated oocysts are stored. Buffers are utilizedin the storage composition as they prolong viability over the use ofsterile water or sterile water containing gentamicin. Many suitablebuffers are known in the art including, but not limited to, phosphatebuffer, bicarbonate buffer, citric acid and tris buffers. In onepreferred embodiment, the diluent comprises 0.5×PBS. In a preferredembodiment, a volume of buffer is used that results in a concentrationof sporulated oocysts suitable for transfer to containers that areultimately used by the consumer as a vaccine for the prevention ofcoccidiosis.

[0189] The diluent may also optionally comprise a bactericide or otherpreservative. Any bactericide that is suitable for use in pharmaceuticalcompositions, and especially compositions that are administered to foodanimals, can be used. Non-limiting examples or bactericides includepotassium perchlorate, sodium hypochlorite, hydrochlorous acid, sodiumhydroxide and antibiotics. Preferred concentrations of chemicalbactericides in final concentration in the vaccine, include: from about0.10 wt % to about 0.25% potassium perchlorate, from about 0.001 wt % toabout 0.01 wt % sodium hypochlorite, from about 1 ppm to about 5 ppmhydrochlorous acid, and from about 0.5 mM to about 1 mM sodiumhydroxide. In another aspect of the instant invention, any antibioticwhich is suitable for incorporation into compositions to be administeredto animals, and especially food animals can be used.

[0190] In one embodiment, after the sterilization and finalconcentration, rather than adding water back to 100% of the volume priorto sterilization in the filtration unit, the volume is brought back upto 50% of the volume prior to sterilization and filtration. In thisparticular embodiment, the tangential flow filter and associated tubingare flushed with a sterile PBS buffer solution to collect any residualsporulated oocysts and the rinse is added to collected retentate. In apreferred embodiment, 1×PBS containing 60 μg/ml gentamicin is added in a1:1 ratio to the disinfected sporulated oocyst suspension to result in asuspension of sporulated oocysts in 0.5×PBS with 30 μg/ml gentamicin. Asample is then taken for assay purposes. Thereafter, the material issubdivided into pre-sterilized containers, sealed, labeled and stored inthe cold room pending filling of containers for commercial distribution,such as vials. This material constitutes a bulk lot.

[0191] In yet another embodiment, the diluent used for storage purposesincludes a composition that ameliorates a decrease in post-challengeperformance and thickening agents to maintain the sporulated oocysts insuspension. Suitable thickening agents include starches, gums,polysaccharides, and mixtures thereof. Suitable compositions toameliorate a decrease in post-challenge performance include, but are notlimited to, cytokines, growth factors, chemokines, mitogens andadjuvants. Such compositions to improve post-challenge performance arewell known to those skilled in the art and can be found, for example, inPlotkin and Orenstein, Vaccines, Third Ed., W. B. Saunders, 1999; Roittet al., Immunology, Fifth Ed., Mosby, 1998; and Brostoff, et al.,Clinical Immunology, Gower Medical Publishing, 1991. Examples ofcompositions to improve post-challenge performance, include, but are notlimited to, Alum (aluminum phosphate or aluminum hydroxide), Freund'sadjuvant, calcium phosphate, beryllium hydroxide, dimethyl dioctadecylammonium bromide, saponins, polyanions, e.g. poly A:U, Quil A, inulin,lipopolysaccharide endotoxins, liposomes, lysolecithins, zymosan,propionibacteria, mycobacteria, and cytokines, such as, interleukin-1,interleukin-2, interleukin-4, interleukin-6, interleukin-12,interferon-α, interferon-γ, granulocyte-colony stimulating factor. Inone preferred embodiment, the diluent includes Propionibacterium acnes(P. acnes) at from between 10 μg and 100 μg per dose (dry weight) andmore preferred at about 50 μg per dose (dry weight). The preferredconcentration is from about 3.0 to about 5.0 milligrams per milliliterof vaccine, most preferably about 4.2 milligrams per milliliter.

[0192] In a further embodiment of the present invention, sterilesporulated oocysts are stored in a composition comprising an oxidizingagent. The oxidizing agent preferably has a reduction potential ofgreater than 0.5 V, more preferably between 0.75 and 3.0 V, mostpreferably between about 1.0 and 2.0 V in the sporulation medium. Theoxygen present in sterile water may also be used as the oxidizing agent.When sterile water, or any other oxidizing agent is used, no additionaloxygen or air is incorporated in to the storage composition. Examples ofother suitable oxidizing agents include, but are not limited to, aqueousbromine, chlorine dioxide, hydrogen peroxide, potassium permanganate,potassium perchlorate, sodium hypochlorite, and hydrochlorous acid whichhave reduction potentials of about 1.09 V, 1.64 V, 1.78 V, 1.49 V, 1.37V, 1.49 V, and 1.63 V, respectively. The requisite amount of oxidizingagent added varies with the agent used and the species of protozoa andcan be determined empirically by one skilled in the art. For protozoa ofthe genus Eimeria, preferred concentrations of the oxidizing agents onceadded to the sporulated oocyst suspension include from about 0.1 toabout 0.75 wt % for potassium perchlorate, from about 0.5 to about 2.9wt % for potassium permanganate, from about 0.001 to about 0.1 wt %sodium hypochlorite and from about 1 ppm to about 5 ppm forhydrochlorous acid.

[0193] In one embodiment, the sporulated oocysts in contact with thediluent retain greater than 60% viability when stored for 13 weeks at25° C. In a preferred embodiment of the instant invention, thesporulated oocysts in contact with the diluent retain greater than 70%viability for at least 26 weeks when stored at 4° C. Because theresulting vaccine is sterile and lacks potassium dichromate, the productis suitable for administration by a variety of routes including, but notlimited to, intravenous, subcutaneous, intramuscular and intraperitonealinjection. Thus, the sporulated oocyst/diluent composition can be usedfor vaccinating animals against coccidiosis.

[0194] The instant invention comprises a composition containing viablesporulated oocysts of a single species or combination of species ofprotozoa known to coccidiosis. The combined species of sporulatedoocysts are present in a number sufficient to comprise the minimumnumber of sporulated oocysts required to comprise an effective dose forimmunizing purposes. The number of sporulated oocysts per dose isfurther determined by the estimated half-life of the sporulated oocystsin the storage composition claimed herein. As the sporulated oocysts agea certain number cease to be functional. A preferred shelf-life isapproximately 12 months. An example of half-life determinations may befound in FIGS. 4 and 5 and Example 4. Therefore, a minimum amount of asingle species or combination of sporulated oocysts is added to thecompositions for consumption that will result in the minimum immunizingdose computed as a function of half-life determinations.

[0195] The storage medium of the current invention contains less thanabout 0.8% by weight of alkali metal dichromate. In more preferredembodiments, the instant invention contains less than about 0.6% byweight of alkali metal dichromate, or less than about 0.4% by weight ofalkali metal dichromate, or less than about 0.2% by weight of alkalimetal dichromate, and less than about 0.1% by weight of alkali metaldichromate.

[0196] Again, as the production of the concentrated vaccine is withoutthe use of an alkali metal dichromate, the storage composition willcontain less than about 0.3% by weight of dichromate ion. In anotherembodiment, the storage composition of the instant invention willcontain less than about 0.15% by weight of hexavalent chromium.

[0197] Furthermore, a most preferred embodiment of the instant inventioncomprises a coccidiosis vaccine for chickens using the sporulatedoocyst/diluent composition of the present invention containing at leastabout 1.5×10⁴ viable wild type sporulated oocysts per milliliter and ischaracterized as substantially free of potassium dichromate.

[0198] In addition, a dye may be added to the vaccine to encourageconsumption. Dyes well known in the art may be used. A preferred dye is0.02% FD+C Emerald Green.

[0199] Vaccine Composition

[0200] The present invention also provides a vaccine composition for theprevention and control of coccidiosis. The vaccine may be concentrated,requiring dilution before administration, or the vaccine may be readyfor administration. The concentrated embodiment of the instant inventionmay be diluted with any suitable diluent to concentrations suitable forvarious forms of administration, including intra-yolk sacadministration, per os, oral gavage, delivery via spray cabinet, ortop-fed via spray on to food, such as OASIS Hatchling Supplement.

[0201] The vaccine composition of the instant invention comprises wildtype sporulated oocysts of at least one species of protozoa known tocause coccidiosis wherein said composition is sterile and contains atleast about 10,000 oocysts per milliliter and less than about 0.8% byweight of alkali metal dichromate. In more preferred embodiments, theinstant invention contains about 10,000 oocysts per milliliter and lessthan about 0.6% by weight of alkali metal dichromate, or about 10,000oocysts per milliliter and less than about 0.4% by weight of alkalimetal dichromate, or about 10,000 oocysts per milliliter and less thanabout 0.2% by weight of alkali metal dichromate, or about 10,000 oocystsper milliliter and less than about 0.1% by weight of alkali metaldichromate. In addition, the vaccine composition of the currentinvention will contain at least about 10,000 oocysts per milliliter andless than about 3.0% by weight of dichromate ion or less than about0.15% by weight of hexavalent chromium.

[0202] In a further embodiment, the concentrated vaccine may be dilutedprior to administration, for example, from 10 milliliters to about a 250milliliter. In another embodiment, the concentrated vaccine may bediluted prior to administration from about 10 milliliters to about 2.5Liters. Such dilute vaccine is sterile and comprises wild type oocystsknown to cause coccidiosis. In a preferred embodiment, said dilutedvaccine composition comprises at least about 1,000 oocysts permilliliter and less than about 0.002% by weight of alkali metaldichromate. In a more preferred embodiment, said diluted vaccinecomprises at least about 1,000 oocysts per milliliter and ischaracterized as substantially free of alkali metal dichromate.

[0203] In a most preferred embodiment, the instant invention is aconcentrated vaccine ready for dilution and then administration whereinsaid concentrated vaccine contains at least about 10,000 sporulatedviable wild type oocysts per milliliter and is characterized assubstantially free of alkali metal dichromate.

[0204] The vaccine composition of the present invention also comprisesviable wild type sporulated oocysts containing less than about 5.0×10⁻³μg of alkali metal dichromate per oocyst. In a preferred embodiment thevaccine composition contains less than about 3.8×10⁻³ μg of alkali metaldichromate per oocyst. In a more preferred embodiment the vaccinecomposition contains less than about 1.3×10⁻³ μg of alkali metaldichromate per oocyst. In a highly preferred embodiment the vaccinecontains less than about 6.3×₁₀ ⁻⁵ μg of alkali metal dichromate peroocyst. In a most preferred embodiment the vaccine composition ischaracterized as substantially free of alkali metal dichromate.

EXAMPLES

[0205] The following examples are intended to provide illustrations ofthe application of the present invention. The following examples are notintended to completely define or otherwise limit the scope of theinvention.

Example 1 Oocyst Collection and Isolation

[0206] Five hundred fifty, 15 day old broiler chickens were infectedwith approximately 7000 viable oocysts per bird of E. tenella by oralgavage or by ingestion via drinking water or feed. Excreta werecollected over a three day period beginning 6 days later at 21 days ofage. Total excreta collected over the three day period was 189 kg.Excreta were processed on the day collected. The excreta collected wereput in a dilution tank maintained at approximately 40-50° F. and dilutedwith water at 0.687 to 0.630 L/bird. The diluted excreta was pumpedthrough a 30″ diameter vibrating sieve fitted with a 50 mesh (297micron) top screen and a 250 mesh (61 micron) bottom screen at a rate ofapproximately 6 LPM. The top two fractions were discarded and thefiltrate, containing the oocysts, was pumped into a chilled (about40-50° F.) collection tank and continuously agitated. The filtrate wasthen pumped at a feed rate of approximately 2.9-3.5 LPM into a SharplesSuper-D-Canter centrifuge. The centrifuge settings were: bowl speed3990-4004 RPM; auger speed 2306-3990 RPM and RPM delta 16.84-17.33. RPMdelta is a measure of the difference between bowl and auger speeds.Total run time ranged from 97 to 100 minutes. The centrate was discardedand the solids (cake), which contained oocysts, were collected into astainless steel tray, weighed and stored in a tank at about 40-50° F.The solids obtained from each of three collection days were combined.

[0207] The volume of solids from the combined three runs was 28 L. Tothese solids was added 25.6 L of high fructose corn syrup and 46.4 L ofwater to give a total volume of 100 L and a specific gravity of 1.094g/l. This material was then centrifuged using a Sharples Super-D-Cantercentrifuge at a bowl speed of 5998 RPM, an auger speed of 3998 RPM and aRPM delta of 20.41. The feed rate was 1.1 l/min and the total run timewas 95 minutes. The centrate, containing the oocysts, was collected andstored in a tank at approximately 40-50° F. and the unwanted excretasolids discarded.

[0208] In order to remove the residual sugar in solution and toconcentrate the oocysts further, the centrate was subjected to anadditional centrifugation. To the 96 L of centrate obtained was added114 L of water to give a final volume of 210 L. The centrifuge settingswere bowl speed 6011 RPM and auger speed 4050 RPM. The initial RPM deltawas 20.01 but was decreased to 15 and then 10 during the run to increasecentrate flow. The centrate was discarded and the solid containing theoocysts was retained. The oocysts were placed in a sterilized containerwith sterile water at a preferred concentration of between about5×10⁶/ml and about 50×10⁶/ml and held at from about 2° C. to about 8° C.until transferred to the sporulation vessel to undergo sporulation.

Example 2 Separation by Hydrocyclone

[0209] The manure from 400 host birds inoculated with E. maxima wascollected from a one day period resulting in 45 kg of manure. Thismanure was diluted and sieved according to the method of Example 1 togive a filtrate volume of 270 L containing 6% solids and 4.64×10⁹oocysts. The filtrate was then introduced to the hydrocyclone by a highpressure pump at a feed rate of approximately 2 gallons per minute andat a pressure of 126 psi.

[0210] The first run resulted in the an upper outlet volume (overflow)of 182 L containing 1.58×10⁸ oocysts and a lower outlet volume of 88 Lcontaining 8% solids and 4.02×10⁹ oocysts. Overflow material wasdiscarded after each run. A second run resulted in an upper outletvolume of 56 L containing 1.46×10⁸ oocysts and a lower outlet volume of28 L containing a 13% solids and 3.25×10⁹ oocysts. A third and final runresulted in an overflow volume of 18 L containing 1.27×10⁸ and a finalvolume for the lower outlet of 10.5 L containing 27% solids and 3.56×10⁹oocysts.

Example 3 Sporulation

[0211] To the oocysts obtained as described in Example 1, was addedenough of a 5.25% sodium hypochlorite solution (CLOROX) to obtain afinal concentration 0.05 wt % sodium hypochlorite. This oocyst/sodiumhypochlorite mixture was added to a 10 liter fermentor set at 28±1° C.and an agitation rate of 200 RPM. Oxygen was provided by portable oxygencylinders and bubbled through the mixture at a rate sufficient to obtaina percent saturation of dissolved oxygen value of at least 50% ofsaturation of dissolved oxygen. Oxygen flow was adjusted so as not tocause foaming of the mixture. The oocysts were maintained under theseconditions for about 72 hours. During sporulation, dissolved oxygen andpH were constantly monitored. It was observed that beginning atapproximately 12 hours into the sporulation process there was a decreasein the percent saturation of dissolved oxygen (increased oxygenconsumption) followed by an increase in pH and a return of dissolvedoxygen to previous levels (FIG. 1). In some, but not all cases, theincrease in pH was preceded by a decrease in pH at about the same timeas the decrease in the percent saturation of dissolved oxygen (FIG. 2).These changes in dissolved oxygen and pH were found to be reliableindicators of sporulation. Examination of oocysts following these changeshowed a high degree of sporulation. In contrast when these changes werenot observed, the sporulation rate was dramatically reduced fromapproximately 90% to approximately 10%. Although sporulation wascomplete at approximately 24 to 36 hours, the incubation was continuedfor another 36 to 48 hours to provide a more stable sporulated oocystpopulation.

[0212] Oocyst quantities for individual species are approximately asfollows: TABLE 2 Spp. of Eimeria Quantity E. acervulina   22 million/mLE. maxima 13.6 million/mL E. tenella 13.7 million/mL

[0213] The average oocyst sporulation ratio determined for individualspecies was as follows: TABLE 3 Percent average Spp. of Eimeriasporulation ratio E. acervulina 80% E. maxima 90% E. tenella 90%

[0214] The average oocyst viability determination for individual specieswas as follows: TABLE 4 Average oocyst Spp. of Eimeria viability E.acervulina    80% E. maxima 70-80% E. tenella    80%

Example 4 Tangential Flow Filtration/Sterilization

[0215] Following sporulation, the sporulated oocysts were concentratedby tangential flow filtration. To begin, the integrity of the filtermembrane was visually observed prior to assembling the CONSEP system.The filter unit was then assembled according to the appropriate standardoperating procedure (“SOP”) as provided in the manufacture's manual.After the system was assembled about 2 to 4 liters of cold domesticwater was run through the system to check for leaks. If leaks werefound, the system would be disassembled and reassembled after checkingfor the source of the leakage. Particular attention was paid toinspecting the gaskets to assure lack of damage to the gaskets and forproper seating.

[0216] The concentrated sporulated oocysts medium was then placed intothe retentate vessel of the filter unit. Domestic cold water was addedto adjust the retentate vessel volume to the desired operating level andalso maintain less than a desired amount of solids. The water source wasthen connected to the retentate vessel through an air-tight fitting.This facilitates operating diafiltration at a constant volume.

[0217] The permeate flow valve was then closed. The control of thediaphragm pump was set to give the desired flow and then the pump wasstarted. The permeate flow valve was then opened after substantially allof the bubbles were removed from the membranes and a steady flow wasestablished across the membrane. The permeate was then directed to aseparate collection container. The permeate sample was collected afterabout 2 to 5 minutes of operation and checked for sporulated oocysts.The sporulated oocysts are to be retained in the system. If sporulatedoocysts were found in the permeate, the filtration would have beenstopped and the source of retentate leakage identified. Retentateleakage often occurs from gaskets around the membrane or when theintegrity of the membrane is compromised. The source of a leak must bedetected and corrected before proceeding with permeation. Permeatecollection, found to be without sporulated oocysts, was then discarded.If sporulated oocysts were found, the permeate would have been returnedto the filter unit to recover any sporulated oocysts that may haveleaked through into the permeate.

[0218] The flow rate of the permeate was checked periodically bymeasuring the volume of permeate by collecting the permeate in agraduated cylinder. The retentate tank volume was maintained at aconstant volume. A small sample of the permeate was collected afterevery 2 liters of permeate was collected from the permeate line and theoptical density at 600 nm (OD₆₀₀) was measured. Once the OD₆₀₀ of thepermeate was less than 0.5, the diafiltration was stopped by closing thepermeate value and disconnecting the water source. The pump's inletlines were then removed from the retentate vessel and connected to aclean water source. The membrane were then flushed with about 500 toabout 1000 ml of water to recover any sporulated oocysts. The retentatevessel was then stored overnight at about 4° C. in a refrigerator. Theretentate was stored overnight to allow the sporulated oocysts to settleto the bottom of the retentate vessel. The layer of retentate over thesettled oocysts was then siphoned off.

[0219] The sporulated oocysts were then sterilized. The filter unit wassterilized by using 5.25% sodium hypochlorite solution to disinfect thesystem. After the sodium hypochlorite was added, all subsequentprocedures were conducted in a HEPA filtered laminar flow hood tomaintain asepsis.

[0220] The Optisep filter unit was assembled according to themanufacture's directions. For E. maxima and E. tenella a 10-micronSpectra/Mesh filter was used. For E. acervulina a 5-micron or a10-micron filter was used. With all species the following procedureswere the same.

[0221] The inlet (retentate return) and outlet tubing (permeate) wereplaced in a beaker containing approximately 400 ml of about 5.25% sodiumhypochlorite. The pump on the filter unit was then started to flush thesystem with the 5.25% sodium hypochlorite. The pump was then stopped andall the valves were closed to let the system equilibrate with the 5.25%sodium hypochlorite in the chamber and tubing for about 15 minutes.

[0222] The sporulated oocyst containing vessel was then removed fromrefrigeration. The supernatant was pumped out without disturbing thesporulated oocyst layer. Enough supernatant was left behind so that thetotal solids were less than approximately 15% by volume. The sporulatedoocyst suspension was then transferred to a retentate vessel.

[0223] An equal volume of about 10% sodium hypochlorite was added to thesporulated oocyst suspension to result in a final concentration ofsodium hypochlorite of approximately 5% and a solids concentration ofless than about 7.5% solids in suspension. The medium was mixedthoroughly and allowed to stand for about 15 minutes.

[0224] The filtration was then begun. Autoclaved water was used as thewater source for filtration. The retentate pump was activated and set at0.6 liters per minute. The permeate line was pinched closed at thistime. Once air bubbles were worked through the filter membrane andtubing the permeate line was opened and directed to a collection vesseloutside the laminar flow hood. The retentate flow was then increased to2 liters per minute. A sample of the permeate was then taken and sampledfor sporulated oocysts. Finding no sporulated oocysts it was determinedthat there was no breach of a membrane or failure of a gasket.

[0225] The volume of the retentate vessel was maintained substantiallyconstant by the addition of either autoclaved or sterilized water. Thefiltration was continued until there was no chlorine odor emanating fromthe permeate. This required about 10 volumes of retentate to run throughthe system. A sample of the permeate was then analyzed for residualchlorine. The filtration was run until the permeate sample containedless than about 1 ppm of chlorine.

[0226] Once the chlorine level was sufficiently reduced, the retentatevolume was then reduced by discontinuing the addition of autoclaved orsterilized water. The concentrated retentate was then asepticallytransferred to a glass vessel wherein an equal volume of 1×PBS with 60μg/ml of gentamicin was added to the disinfected sporulated oocystssuspension. This resulted in a suspension of sporulated oocysts in0.5×PBS with approximately 30 μg/ml gentamicin. The solution was thenplaced in refrigeration at approximately 4° C. for future use.

Example 5 Storage

[0227] Sterile sporulated oocysts were stored in 0.1% potassiumperchlorate, 0.001% sodium hypochlorite, reverse osmosis/deionized(RO/DI) water or 0.5×PBS containing 30 μg/ml gentamicin and at either 4°C. or room temperature (25° C.). In some instances the storage mediumalso contained Propionibacterium acnes at a concentration of 10-100μg/dose (dry weight). As used herein, a dose is the amount to beadministered to an individual animal at one time. At the times indicatedin FIGS. 3 and 4, samples were aseptically removed from the storagecontainers and tested for viability by vital staining.

[0228] The results are shown in FIGS. 4 and 5. The method ofsterilization, either 2% or 5% sodium hypochlorite did not appear tohave a significant effect on viability during storage. Sporulatedoocysts which had not been sterilized, however, showed a rapid decreasein viability when stored.

[0229] Storage temperature was found to have an effect on ability ofsporulated oocysts to remain viable during storage. Sporulated oocystsmaintained at 4° C. maintained their viability for at least 26 weekswhen stored in any of the medium tested. Sporulated oocysts stored atroom temperature, however, showed a marked decrease in viability by 20weeks in storage. When stored at 4° C., all groups of sporulated oocystsmaintained at least 70% viability over the 26 week test period. In termsof change in percent viable oocysts recovered (PVOR), a comparison ofPVOR at the first and last sampling periods shows that in no case didthe decrease in PVOR exceed 10% when stored at 4° C. These results showthat it is possible to maintain sporulated oocysts for extended periodsof time in sterile medium lacking potassium dichromate without asignificant loss in viability.

[0230] In light of the detailed description of the invention and theexamples presented above, it can be appreciated that the several aspectsof the invention are achieved.

[0231] It is to be understood that the present invention has beendescribed in detail by way of illustration and example in order toacquaint others skilled in the art with the invention, its principles,and its practical application. Particular formulations and processes ofthe present invention are not limited to the descriptions of thespecific embodiments presented, but rather the descriptions and examplesshould be viewed in terms of the claims that follow and theirequivalents. While some of the examples and descriptions above includesome conclusions about the way the invention may function, the inventorsdo not intend to be bound by those conclusions and functions, but putthem forth only as possible explanations.

[0232] It is to be further understood that the specific embodiments ofthe present invention as set forth are not intended as being exhaustiveor limiting of the invention, and that many alternatives, modifications,and variations will be apparent to those of ordinary skill in the art inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and scope ofthe following claims.

What is claimed is:
 1. A method for producing a composition for theprevention or control of coccidiosis comprising: collecting manure fromhost animals wherein said manure contains oocysts known to causecoccidiosis; diluting said manure in an aqueous medium to create aslurry; separating unwanted fecal matter from said slurry and collectingthe aqueous fraction containing oocysts; subjecting said aqueousfraction to solid/liquid phase centrifugal-based separation andcollecting the solid phase; combining a dense aqueous liquid with saidcollected solid phase wherein said dense liquid has a density greaterthan about 1.09 g/ml and wherein the oocysts are buoyant; subjecting thecombination of said dense aqueous liquid and collected solid phase tocentrifugation and collecting the dense liquid fraction containingoocysts; diluting said dense liquid fraction to a specific gravitywherein the oocysts are no longer buoyant; separating oocyst solids fromsaid diluted liquid fraction by centrifugal-based separation andre-collecting the solid phase.
 2. A method as set forth in claim 1further comprising: diluting said re-collected solid phase in an aqueoussporulation medium; sporulating said oocysts while in contact with saidsporulation medium; separating sporulated oocysts from said sporulationmedium; sterilizing said sporulated oocysts; and diluting saidsporulated oocysts to form a vaccine composition.
 3. A method ofseparating oocysts from a liquid suspension by the use of ahydrocyclone.
 4. A method as set forth in claim 3 wherein the oocystsare collected in the underflow from the hydrocyclone.
 5. A method forisolating oocysts comprising: collecting manure from host animalswherein said manure contains oocysts known to cause coccidiosis;diluting said manure in an aqueous medium to create a slurry; separatingunwanted fecal matter from said slurry and collecting the aqueousfraction containing oocysts; subjecting said aqueous fraction tosolid/liquid phase centrifugal-based separation by means of ahydrocyclone.
 6. A method for isolating oocysts comprising: collectingmanure from host animals wherein said manure contains oocysts known tocause coccidiosis; diluting said manure in an aqueous medium to create aslurry; separating unwanted fecal matter from said slurry and collectingthe aqueous fraction containing oocysts; subjecting said aqueousfraction to solid/liquid phase centrifugal-based separation andcollecting the solid phase; combining a dense aqueous liquid with saidcollected solid phase wherein said dense liquid has a density greaterthan about 1.09 g/ml and wherein the oocysts are buoyant; subjecting thecombination of said dense aqueous liquid and collected solid phase tocentrifugation and collecting the dense liquid fraction containingoocysts; diluting said dense liquid fraction to a specific gravitywherein the oocysts are no longer buoyant; separating oocyst solids fromsaid liquid phase by means of a hydrocyclone and re-collecting the solidphase.
 7. A method for isolating oocysts comprising: collecting fecesfrom animals wherein said feces contains oocysts known to causecoccidiosis; contacting said feces with an aqueous medium; separatingunwanted fecal matter from said oocysts; subjecting said oocysts tocentrifugal-based separation and collecting the oocyst-containing solidfraction; suspending the oocyst-containing solid fraction in a flotationsolution; allowing the oocysts to separate from the solids, wherein theoocysts are floated to the top of the solution; and removing theflotation medium from said oocysts by tangential flow filtration.
 8. Amethod as set forth in claim 1 wherein said animals comprise the classAves.
 9. A method as set forth in claim 8 wherein said slurry is createdby mixing manure and water in relative proportions of from about 0.5gallons to about 5 gallons of domestic water per the amount of manureobtained in about 3 days from about six animals comprising the classAves.
 10. A method as set forth in claim 9 wherein said animals arechickens.
 11. A method as set forth in claim 1 wherein said separationof unwanted fecal matter comprises sieving.
 12. A method as set forth inclaim 11 wherein said sieving is by the use of multiple-tier shakerscreens.
 13. A method as set forth in claim 12 wherein said shakerscreens comprise a 50-mesh screen and a 250-mesh screen.
 14. A method asset forth in claim 1 wherein said method is carried out at a temperaturebetween about 4° C. and about 30° C.
 15. A method as set forth in claim14 wherein said sieving is carried out at a temperature between about22° C. and about 28° C.
 16. A method as set forth in claim 15 whereinsaid sieving is carried out at about 25° C.
 17. A method as set forth inclaim 1 wherein each of said centrifugal-based separations comprises theuse of a centrifuge or a hydrocylcone.
 18. A method as set forth inclaim 17 wherein said centrifugal-based separation comprises the use ofa hydrocyclone.
 19. A method as set forth in claim 18 wherein saidcentrifugal-based separation comprises the use of a centrifuge.
 20. Amethod as set forth in claim 19 wherein said centrifuge is a bottlecentrifuge.
 21. A method as set forth in claim 19 wherein saidcentrifuge is a continuous centrifuge.
 22. A method as set forth inclaim 1 wherein said centrifugation is a bottle centrifuge.
 23. A methodas set forth in claim 1 wherein said dense aqueous liquid comprises asolution of corn syrup or sodium chloride.
 24. A method as set forth inclaim 1 wherein said aqueous solution has a density from about 1.07 g/mlto about 1.20 g/ml.
 25. A method as set forth in claim 24 wherein saidaqueous solution has a density from about 1.08 g/ml to about 1.14 g/ml.26. A method as set forth in claim 25 wherein said aqueous solution hasa density from about 1.09 g/ml to about 1.10 g/ml.
 27. A method forinducing sporulation of oocysts comprising: introducing into an aqueoussporulation medium oocysts of at least one species of protozoa known tocause coccidiosis; incubating said oocysts in said aqueous sporulationmedium, thereby causing sporulation of oocysts; and introducing anoxidizing agent into said medium at a rate sufficient to maintain theaverage dissolved oxygen content during sporulation at at least 30% ofsaturation.
 28. A method as set forth in claim 27 wherein said dissolvedoxygen content is substantially maintained at between about 30% andabout 80% of saturation throughout the period of sporulation.
 29. Amethod as set forth in claim 28 wherein said dissolved oxygen content ofthe medium is substantially maintained at between about 40% and about60% of saturation throughout the period of sporulation.
 30. A method asset forth in claim 29 wherein said dissolved oxygen content of themedium is substantially maintained at about 50% of saturation.
 31. Amethod as set forth in claim 27 wherein the alkali metal dichromatecontent of said sporulation medium is less than about 0.8% by weightduring incubation of oocysts.
 32. A method as set forth in claim 27comprising addition to said sporulation medium of an oxidizing agenthaving a standard reduction potential of at least about 0.5 V.
 33. Amethod as set forth in claim 32 comprising addition of both molecularoxygen and another oxidizing agent.
 34. A method as set forth in claim33 wherein said oxidizing agent has a standard reduction potential of atleast about 0.5 V.
 35. A method as set forth in claim 34 wherein saidoxidizing agent is selected from the group consisting of an alkali metalhypochlorite, an alkali metal chlorite, an alkali metal chlorate, analkali metal perchlorate, and an alkali metal permanganate.
 36. A methodas set forth in claim 35 wherein said oxidizing agent compriseshypochlorite ions.
 37. A method as set forth in claim 35 wherein asufficient amount of an alkali metal hypochlorite is added to achieve analkali metal hypochlorite weight percent from about 0.001 weight percentto about 0.1 weight percent of the sporulation medium and oocystscombined, wherein said alkali metal hypochlorite is from about 1.0% toabout 10.0% by volume.
 38. A method as set forth in claim 27 furthercomprising: separating sporulated oocysts from said sporulation medium;sterilizing sporulated oocysts by contacting said sporulated oocystswith a chemical disinfectant; and storing said sporulated oocysts in asterile diluent, said diluent containing less than about 0.8% by weightalkali metal dichromate.
 39. A method as set forth in claim 27 whereinsaid medium contains less than about 0.3% by weight dichromate ionduring incubation of said oocysts.
 40. A method as set forth in claim 27wherein said medium contains less than about 0.15% by weight hexavalentchromium during incubation of said oocysts.
 41. A method as set forth inclaim 27 wherein said dissolved oxygen content is established bybubbling an oxygen-containing gas through said sporulation medium.
 42. Amethod as set forth in claim 41 wherein said oxygen-containing gasconsists essentially of air.
 43. A method as set forth in claim 41wherein said gas comprises commercially pure oxygen.
 44. A method as setforth in claim 27 further comprising maintaining the temperature from atemperature that substantially avoids freezing to about 45° C.
 45. Amethod as set forth in claim 44 wherein temperature is maintained fromabout 15° C. to about 40° C.
 46. A method as set forth in claim 45wherein temperature is maintained from about 20° C. to about 30° C. 47.A method as set forth in claim 46 wherein temperature is maintained atabout 28° C.
 48. A method as set forth in claim 27 further comprisingincubating the oocysts under said conditions from about 72 hours toabout 120 hours.
 49. A method as set forth in claim 48 wherein theoocysts incubate from about 72 hours to about 96 hours.
 50. A method asset forth in claim 49 wherein the oocysts incubate for about 72 hours.51. A method as set forth in claim 27 further comprising controlling thepH of the sporulation medium.
 52. A method as set forth in claim 51wherein the pH is controlled by the introduction of an acid or base tothe sporulation medium.
 53. A method as set forth in claim 52 whereinthe pH of the sporulation medium is controlled by alternatively addingsodium hydroxide and sulfuric acid to the sporulation medium.
 54. Amethod as set forth in claim 53 wherein the pH of the sporulation mediumis controlled from about 7.2 to about 7.5.
 55. A method as set forth inclaim 54 wherein the pH of the sporulation medium is controlled at aboutfrom 7.35 to about 7.45.
 56. A method as set forth in claim 38 whereinsaid sporulated oocysts are separated from said sporulation medium byfiltration or by centrifugal-based separation.
 57. A method as set forthin claim 56 wherein said sporulated oocysts are separated by filtration.58. A method as set forth in claim 57 wherein said sporulated oocystsare separated from the sporulation medium by tangential flow filtration.59. A method as set forth in claim 38 wherein said sterilization isachieved by adding a chemical disinfectant to sporulated oocystsseparated from said sporulation medium.
 60. A method as set forth inclaim 59 wherein said sterilization substantially eliminatesmicroorganisms.
 61. A method as set forth in claim 60 wherein saidmicroorganisms are selected from the group comprising infectious bursaldisease virus and chicken anemia virus.
 62. A method as set forth inclaim 59 wherein said sterilization is by a chemical disinfectant otherthan an alkali metal dichromate.
 63. A method as set forth in claim 59wherein said chemical disinfectant comprises a solution of an alkalimetal hypochlorite.
 64. A method as set forth in claim 63 wherein saidchemical disinfectant comprises a solution of sodium hypochlorite.
 65. Amethod as set forth in claim 64 wherein said solution used is at aconcentration from about 1% to about 20% by volume of active chlorine.66. A method as set forth in claim 65 wherein said is at a concentrationfrom about 5% to about 15% by volume of active chlorine.
 67. A method asset forth in claim 66 wherein said solution is at a concentration ofabout 10% by volume of active chlorine.
 68. A method as set forth inclaim 64 wherein said sporulated oocysts are treated with said sodiumhypochlorite from about 5 to about 25 minutes.
 69. A method as set forthin claim 68 wherein said sporulated oocysts are treated with said sodiumhypochlorite from about 8 to about 20 minutes.
 70. A method as set forthin claim 69 wherein said sporulated oocysts are treated with said sodiumhypochlorite for about 10 minutes.
 71. A method as set forth in 63further comprising substantially separating said sodium hypochloritefrom the sporulated oocysts by filtration.
 72. A method as set forth inclaim 71 wherein said filtration is by means of tangential flowfiltration.
 73. A method for inducing sporulation of oocysts comprising:introducing into an aqueous sporulation medium oocysts of at least onespecies of protozoa known to cause coccidiosis; incubating said oocystsin said aqueous sporulation medium, thereby causing sporulation ofoocysts; and introducing an oxidizing agent having a standard reductionpotential of at least about 0.5 V into said medium at a rate sufficientto maintain the oxidation potential of said medium equivalent to theoxidation potential of a medium containing dissolved molecular oxygen inconcentration of at least 30% of saturation during sporulation; saidmedium containing less than about 0.8% by weight alkali metal dichromateduring incubation of said oocysts.
 74. A method for sporulating oocystscomprising: introducing into an aqueous sporulation medium oocysts of atleast one species of protozoa known to cause coccidiosis; incubatingsaid oocysts in said aqueous sporulation medium, thereby causingsporulation of oocysts; and separating said oocysts by tangential flowfiltration from said sporulation medium.
 75. A method for sterilizingoocysts comprising: contacting oocysts of at least one species ofprotozoa known to cause coccidiosis with a sterilization medium; andremoving said sterilization medium from said oocysts by tangential flowfiltration.
 76. A method for monitoring sporulation of oocystscomprising: incubating viable oocysts in an aqueous sporulation medium;and during incubation, monitoring said medium to detect a change in atleast one of the following parameters: (i) dissolved oxygen content;(ii) pH; (iii) rate of introduction of oxidizing agent into said medium;(iv) flow rate of acid or base into said medium.
 77. A method as setforth in claim 76 wherein dissolved oxygen content of said medium iscontrolled by addition of molecular oxygen thereto, and monitoringsporulation comprises detecting a change in oxygen consumption asindicated by detection of a change in oxygen flow to the medium and/or apermanent or transient change in dissolved oxygen content.
 78. A methodas set forth in claim 76 wherein pH of said medium is controlled byaddition of acid or base thereto, and monitoring sporulation comprisesdetecting an increase in acid consumption as indicated by an increase inacid flow to the medium and/or a permanent or transient increase in pH.79. A method as set forth in claim 76 wherein the end point ofsporulation is determined from substantial cessation of oxygenconsumption or generation of alkalinity in the sporulation medium.
 80. Amethod as set forth in claim 79 wherein the end point is indicated bythe substantial cessation of change in at least one of said parameters.81. A method as set forth in claim 79 wherein said sporulated oocystsare maintained is said medium under sporulation conditions for at leastanother 48 hours after the indicated end point of sporulation.
 82. Amethod as set forth in claim 76 wherein said change in dissolved oxygencontent is a decrease.
 83. A method as set forth in claim 76 whereinsaid change in pH is an increase.
 84. A method as set forth in claim 83wherein said increase in pH is at least 0.5 pH units.
 85. A method asset forth in claim 84 wherein said increase in pH is at least 0.25 pHunits.
 86. A composition for the storage of sporulated oocystscomprising an aqueous diluent and a bactericide, said compositioncharacterized as substantially free of alkali metal dichromate whereinsaid composition is characterized as having: a diluent comprising 0.5×phosphate buffered saline; a pH from about 5.0 to about 8.0; and whereinsaid bactericide is selected from the group consisting of an alkalimetal perchlorate, an alkali metal hypochlorite, hydrochlorous acid,sodium hydroxide and antibiotics.
 87. A composition as set forth inclaim 86 having a pH from about 7.0 to about 7.5.
 88. A composition asset forth in claim 86 wherein said bactericide comprises gentamicin. 89.A composition as set forth in claim 86 further comprising an oxidizingagent.
 90. A composition as set forth in claim 86 characterized in thatan oocyst population in contact with said composition remains at leastabout 60% viable for 13 weeks at about 25° C.
 91. A composition as setforth in claim 86 characterized in that an oocyst population in contactwith said composition remains at least about 60% viable for 26 weeks atabout 5° C.
 92. A composition as set forth in claim 86 characterized inthat an oocyst population in contact with said composition decrease inviability no more than about 20% over a period of at least about 13weeks at about 25° C.
 93. A composition as set forth in claim 86characterized in that an oocyst population in contact with saidcomposition decrease in viability no more than about 20% over a periodof at least about 26 weeks at about 5° C.
 94. A composition as set forthin claim 86 further comprising a dye.
 95. A composition for the storageof sporulated oocysts comprising: 0.5×PBS; and about 30 μg/mlgentamicin, said composition characterized as substantially free ofalkali metal dichromate, and wherein said composition is characterizedin that oocysts in contact with said composition decrease in viabilityno more than about 20% over a period of at least about 26 weeks at about5° C.
 96. A method for storing sporulated oocysts comprising contactingsaid sporulated oocysts with the composition of claim
 86. 97. A methodas set forth in claim 96 further comprising storing said sporulatedoocysts in contact with the composition of claim 86 at either about 25°C. or about 5° C.
 98. A method as set forth in claim 96 wherein saidpopulation of sporulated oocysts is maintained at least 60% viable for13 weeks at about 26° C.
 99. A method as set forth in claim 96 whereinsaid population of sporulated oocysts is maintained at least 60%viability for 26 weeks at about 5° C.
 100. A method as set forth inclaim 96 wherein said method prevents a decrease in oocyst viability ofgreater than 20% over a period of at least 13 weeks at about 25° C. 101.A method as set forth in claim 96 wherein said method prevents adecrease in viability of greater than 20% in a population of sporulatedoocysts over a period of at least 26 weeks at about 5° C.